Antibiotic resistance of Bacteroides and Anaerostipes isolates from feces of ICU hospitalized patients

Chantal Deen

Msc Biotechnology

910203 174 010

Course code: MIB-80424

Supervisor: Teresita Bello Gonzalez

Examinator: Hauke Smidt

December 2014, Wageningen University and Research

-Antibiotic resistance of Bacteroides and Anaerostipes isolates from feces of ICU hospitalized patients-

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Table of Content:

List of tables...........................................................................................................................................5

List of figures..........................................................................................................................................7

Acknowledgements................................................................................................................................9

Abstract................................................................................................................................................10

Introduction.....................................................................................................................................11

1. Composition of microbiota in human gut.................................................................................11

1.1. Bacteroides...........................................................................................................................12

1.2. Anaerostipes.........................................................................................................................15

1.3. Antibiotics.............................................................................................................................16

1.4. Antibiotic treatments at intensive care: SOD vs SSD............................................................20

1.5. Effect of antibiotics on the microbial community in the gut.................................................21

1.6. Mechanisms of antibiotic resistance....................................................................................22

1.7. Mechanisms of genetic transfer...........................................................................................25

1.8. Antibiotic resistance in anaerobes: Bacteroides...................................................................29

1.9. Research questions and approach........................................................................................30

2. Materials and methods................................................................................................................31

2.1. Isolation of bacterial species from feacal samples................................................................31

2.2. Identification of patient samples by sequencing, blasting and building a phylogenetic tree33

2.3. Physiological and biochemical characterisation of Bacteroides and Anaerostipes...............34

2.4. Predict antibiotic resistance with ARDB................................................................................35

Techniques to determine antibiotic resistance profile.....................................................................36

2.5. Testing transformation of antibiotic resistance genes..........................................................40

2.6. Agarose gel electrophoresis.................................................................................................41

2.7. PCR erm Genes.....................................................................................................................41

3. Results..........................................................................................................................................42

3.1. Re-grown and obtain single colonies....................................................................................42

3.2. API test Anaerostipes............................................................................................................48

3.3. Antibiotic resistance profile..................................................................................................50

3.4. Transformation experiments................................................................................................58

4. Final discussion and conclusion....................................................................................................62

4.5. Plasmid transformation of E.coli...........................................................................................70

4.6. Conclusions: Clinical failures involving Bacteroides..............................................................70

4.7. Trouble shooting in general..................................................................................................73

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References............................................................................................................................................74

5. Appendix......................................................................................................................................79

5.1. Anaerobic culturing techniques of Bacteroides....................................................................79

5.2. Anaerobic culturing techniques of Anaerostipes..................................................................79

5.3. Gram staining protocol.........................................................................................................80

5.4. DNA extraction.....................................................................................................................80

5.5. DNA purification...................................................................................................................80

5.6. Motility testing.....................................................................................................................80

5.7. API test Anaerostipes............................................................................................................80

5.8. Results disc diffusion test: Pictures Anaerostipes.................................................................84

5.9. Pictures of the disc diffusion test: Bacteroides.....................................................................86

5.10. Preparing non-selective (Mueller Hinton) medium for antibiotic resistant profiling........87

5.11. Preparing non-selective (Wilkins Chalgren) medium for antibiotic resistant profiling......87

5.12. 16S rRNA gene PCR...........................................................................................................87

5.13. Colony PCR........................................................................................................................88

5.14. Preparing McFarland Standards.......................................................................................89

5.15. Phosphate buffers.............................................................................................................89

5.16. Agar and broth dilution method.......................................................................................90

5.17. β-lactamase disk test........................................................................................................90

5.18. CCMB80 buffer.................................................................................................................91

5.19. Preparing competent cells................................................................................................91

5.20. Transformation.................................................................................................................92

5.21. LB (agar) medium..............................................................................................................92

5.22. SOB and SOC (agar) medium............................................................................................92

5.23. Plasmid isolation...............................................................................................................93

5.24. Plasmid digestion with Alu1..............................................................................................93

5.25. Agarose gel electrophoresis..............................................................................................93

5.26. PCR Erm genes..................................................................................................................94

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List of tablesTable 1. The table shows patient nr, time point (days), growth medium and antibiotics used to isolate the

different Bacteroides strains................................................................................................................ 31Table 2. Antibiotic resistance tested for the Anaerostipes isolates................................................................34Table 3. Antibiotic resistance tested for Bacteroides isolates........................................................................34Table 4. Antibiotics tested for agar diffusion tests. These agar diffusion tests are divided in three different

experiments: agar diffusion, double disk test and β-lactamase test......................................................35Table 5. Discs and antibiotics used to determine β-lactamase resistance in Bacteriodes................................35Table 6. Phenotypes of the beta-lactamase test...........................................................................................36Table 7. Antibiotics tested during double disk test.......................................................................................37Table 9. Isolates from patient 120 identified as Anaerostipes.......................................................................42Table 10. Isolates from patient 120 identified as Bacteroides, Parabacteroides and Odoribacter...................43Table 11. The isolates and growth conditions and the identification done with NCBI and with RDP, showing

the name of the species and accession number in the database...........................................................44Table 12. Results of the API test for Anaerostipes.........................................................................................47Table 13. Identification Anaerostipes isolates from patients faecal...............................................................48Table 13. Agar dilution test of the individual isolates...................................................................................49Table 14. The results of the disc diffusion test of each isolate.......................................................................51Table 15. β-lactamase disc test of Bacteroides..............................................................................................53Table 17. Double disc test performed on Anaerostipes isolates....................................................................55Table 17. E-test performed with Anaerostipes resistant to vancomycin........................................................56Table 18. Plasmids isolated from the isolates able to grow on antibiotics.....................................................57Table 20. Antibiotic resistant profile of E.coli................................................................................................60Table 20. Summary of the results of the susceptibility tests done of all the isolates......................................63Table 22. BM liquid medium........................................................................................................................ 78Table 23. Haemin and VitK1 added to BM, RCA and MH medium..................................................................78Table 24. Components for RCA medium....................................................................................................... 78Table 25. Results of the API test for Anaerostipes.........................................................................................81Table 26. Ingredients MH medium per litre..................................................................................................86Table 27. Ingredients WC medium per litre..................................................................................................86Table 28. Components of PCR mastermix.....................................................................................................86Table 29. PCR programme used for 16S rRNA PCR........................................................................................87Table 30. Mastermix prepared per PCR reaction...........................................................................................87Table 31. Programme used for amplification of the DNA..............................................................................87Table 32. Dilutions made for McFarland standards.......................................................................................88Table 33. antibiotics used for agar diffusion method....................................................................................89Table 34. Antibiotics tested during the β-lactamase disk test........................................................................89Table 35. Interpretation of the β-lactamase disc test....................................................................................90Table 36. components needed for CCMB80 buffer........................................................................................90Table 37. Below shows the components and quantities needed for LB medium............................................91Table 38. Components needed for SOB and SOC agar medium.....................................................................91Table 39. Mixture made for restriction enzyme analysis...............................................................................92Table 40. mastermix prepared per gene.......................................................................................................93Table 41. Steps done in the PCR programme................................................................................................93

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List of figuresFigure 1. The composition of the intestines..................................................................................................12Figure 2. Phylogenetic tree of the members of the Bacteroides fragilis group. The tree is based on 16s rRNA

genes and the bootstrap value is 500 [20]............................................................................................13Figure 3. Phylogenetic tree from Anaerostipes species based on 16S rRNA sequences and the numbers in the

figure are bootstrap values n=500........................................................................................................15Figure 4. The effects of antibiotic treatment on the composition of microbiota............................................21Figure 5. Antibiotic resistance mechanisms developed by bacteria; A) efflux pump, B) transfer of antibiotic

resistant genes by plasmids, C) Altering enzymes targeted by the antibiotics, D) development of enzymes that degrade the antibiotic....................................................................................................22

Figure 6. Mechanism of efflux pumps present in the bacterial cell membrane..............................................24Figure 7. Gene transfer between two bacterial cells (schematic)...................................................................26Figure 8. Time scheme (in days) used to collect faecal samples per patient treated with antibiotics. Patients

120 received SSD treatment and patient I SOD treatment. Samples were taken at the start of the treatment, during and after the treatment...........................................................................................31

Figure 9. Scheme used for β-lactam disk test. The order of the antibiotics on the plate and concentration of the discs used are indicated................................................................................................................. 37

Figure 10. Different possible phenotypes in case of erythromycin resistance................................................38Figure 11. Example of E-test......................................................................................................................... 39Figure 12. GeneRuler DNA ladders used on agarose gels...............................................................................41Figure 13. Gram stainings of the different bacterial strains. Bacterial strains identified were Anaerostipes

rhamnosivorans 1y-2, Anaerostipes hadrus sp. 5.1.63FAA, Bacteroides dorei, Bacteroides thetaiotaomicron, Parabacteroides distasonis and Odoribacter splanchnicus.......................................42

Figure 14. Phylogenetic tree of isolates identified as Anaerostipes...............................................................46Figure 15. Phylogenetic tree of the patients isolates. These isolates were identified as Bacteroides,

Parabacteroides or Odoribacter. Bootstrap values is 500.....................................................................47Figure 16. Pictures of the different Bacteroides isolates of which β-lactamase tests were performed............55Figure 17. Double disc showing the M-phenotype of isolate 120D FAA AT 1 (Anaerostipes rhamnosivorans).57Figure 18. Plasmids isolated from Bacteroides and Anaerostipes..................................................................59Figure 19. Agarose gel of the original plasmids from Bacteroides and the plasmids transferred into E.coli.. . .60Figure 20. Bacterial species isolated from each time point per patients........................................................62Figure 21. Spread of antibiotic resistant genes throughout the environment................................................72Figure 22.Pictures taken of API test Anaerostipes. The phenotype is shown and isolate number is given..... .82Figure 23. Pictures of the agar diffusion test of each isolate.........................................................................84Figure 24. Disc diffusions test performed with Anaerostipes isolates showing the inhibition zones...............85Figure 25. Bacteroides isolates were tested with disc diffusion test..............................................................86Figure 26. Scheme of order of the β-lactamase on the medium....................................................................91

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AcknowledgementsI would like to thank Teresita Bello Gonzalez for supervising me during this project. I also would like to thank Dio Ramondrama for helping me find my way in the laboraty. I also could like to thank the technicians and people who wanted the answer my questions and helping me out during this project. And last I want to thank the people from the Microbiology department for all the delicious cookies, cakes and other treats.

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AbstractThe microbiota in the gut has as main function to digest food into nutrients, which were otherwise non-digestible for the host. However, several factors influence the health and composition of the microbial gut, of which antibiotic treatment has the greatest influence. ICU hospitalized patients receive antibiotic treatment to prevent infection, but the drug of choice were designed to minimise the effect on the microbial gut. However, the exact effect of these antibiotic treatments on gut microbes is not known. Although, antibiotic treatment should not influence the composition of the gut, it is suspected that anaerobic bacteria in the gut are a reservoir of antibiotic resistant genes.

During this project anaerobic bacteria were isolated from faecal samples from ICU patients which received SSD or SOD treatment during their hospitalization. Bacteria were characterized by sequencing, Gram stain and sugar test (API 50CH). After identification of the bacteria were antibiotic resistance profile made from the different isolates, based on data obtained from ARDB and from literature sources. Finally was tested if antibiotic resistance was present on plasmids and if these plasmids could be successfully transferred into E.coli cells.

Characterisation led to identification of two bacterial groups; Anaerostipes rhamnosivorans and Bacteroides. Bacteroides species identified were B.dorei and B.thetaiotaomicron. Other bacteria identified were Odoribacter splanchnicus and Parabacteroides distansonis. For Anaerostipes, resistant phenotypes were found for tetracycline and this result was consistent with the database, as Anaerostipes can possess a tetracycline resistance gene called tetO. A resistant gene for bacitracin (Baca) could also be found in Anaerostipes, but this was the case for partly of these bacteria only. Resistance for all Anaerostipes was also found for erythromycin, kanamycin, streptomycin and might by caused by the presence of efflux pumps in the cell membranes, as anaerobic bacteria are known to posses these pumps. Resistance for cefoxitin might be caused by three mechanisms; production of inactivation enzymes or low affinity binding proteins or decreased cell memebrane permeability by loss of porin.

For Bacteroides species, resistance has been expected to tetracyclin, cefoxitin, erythromycin, kanamycin, streptomycin and ampicillin and resistance has been seen for tetracyclin (tetQ), cefoxitin (bl2e cfxa), kanamycin (aphiii3) and erythromycin (ermf). The resistance seen can be described to several genes and mechanisms, which has been identified in Bacteroides before.

The phenotype of resistance is not in line with the antibiotic treatment the patients received. During treatment the patients received cephalosporins and the bacteria tested were indeed resistance to cephalosporins (cefoxitin). But resistance to the other antibiotics tested cannot be explained by the treatment the patients received.

Resistance from to other antibiotic might have been gained by horizontal gene transfer between bacteria who are temporarily present in the gut and between the permanent inhabitants in the gut. Gene transfer has been tested and the transformation was successful for two plasmids. And after transformation the E.coli strains were able to express the plasmid, leading to the expected antibiotic resistance. Concluding, anaerobic bacteria present in the gut are important reservoirs of antibiotic resistance genes.

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IntroductionHumans have co-evolved with a number of microbes. These microbes maintain a commensal or symbiotic relationship with the host [1, 2]. The largest of these microbial population is located in the gastrointestinal tract (GIT) or gut and the majority of these bacteria are anaerobic [1, 3]. Different factors influence the composition of the microbiota and abundance of the bacteria described above. Factors like diet, gender, age and antibiotics can change the microbial community temporarily or permanent and indirectly influence the health of the host [1, 4, 5].

1. Composition of microbiota in human gutThe microbial composition of an individual adult consists of an estimated 10^14 prokaryotic cells belonging to 150-800 species [1]. Bacteria in the human gut are 99% obligate anaerobic of which the major phyla are Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria [6]. These bacteria are common present in human individuals. However, the microbial composition between individuals can be differ greatly and depends on factors like age, diet and lifestyle [1].

After birth, Bacteroides and Bifidobacterium have been found in the stool of infants after roughly ten days, although their numbers are lower when the infant is breast fed. In addition the gut will be invaded in the first days by enterobacteria, streptococci, enterococci, staphylococci and lactobacilli [7]. In general can be said that the composition is in eraly life stage is very diverse and no pre-dominant bacteria can be determined [7].

When an individual ages, this affects the composition of the gut as well [7-9]. Studies have shown that during aging the diversity of the microbial community decreases and also the rate between different bacteria groups changes, sometimes affecting the health of the host. In more detail, the composition of the gut in later stage of life was more enriched with potential pathogens, like Bacteroidetes and was depleted with Firmicutes [8, 9]. Firmicutes are bacteria that produce short chain fatty acid and have therefore a beneficial effect on the host [8, 10, 11]. Other bacteria found more abundant in elderly were Parabacteroides, Eubacterium, Anaerotruncus, Lactonifactor and Coprobacillus and these bacteria are considered as both commensal bacteria and potentially pathogenic [12, 13].

The potential effect of present pathogens on the intestinal cells is shown in Figure 1. The first picture shows the normal situation (figure 1A), and the second picture shows that invasion of the intestinal cells by potential pathogens led to an inflammatory response (figure 1B). If the invading organism is also one of the commensal bacteria this might trigger an inflammatory response against that bacteria and thereby decreasing the level of tolerance in the gut [5, 8, 9, 12].

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Figure 1. Figure 1A shows the composition of the intestines in a healthy situation. The cells are protected by a mucus layer, maintaining the barrier between host and microbes. In figure 1B the mucus layer and host cells has been penetrated by these microbes, leading to an inflammatory response towards these microbes. In case these invaded microbes are normally commensals, this might trigger a lower level of tolerance against these commensals. Figure is adapted from Canny, 2008 [12].

During this study the focus will be on two habitants of the gut microbiota, which are Bacteroides and Anaerostipes.

1.1. BacteroidesBacteroides are a dominant species in the human gut, as it is estimated that Bacteroides together with Parabacteroides species represent about 25% of the intestinal microbiota [14, 15]. These anaerobic, Gram-negative, non-spore forming bacteria have been associated as both commensals and pathogens [12, 16, 17]. As commensals, Bacteroides can ferment a wide range of simple and complex carbohydrates into short chain fatty acids, an energy source that is used by the host which also induce anti-inflammatory responses [12, 13]. One group of Bacteroides, that are known as potential pathogens are members of the B. fragilis group, which is more explained into detail below [18].

1.1.1. Bacteroides fragilis groupMembers of the Bacteroides fragilis group have been associated with development in inflammation [12]. Patients suffering from inflammatory bowel disease have an increased abundance of B. fragilis in the gut, when compared to other Bacteroides present [12, 13]. As pathogens Bacteroides fragilis cause severe intra-abdominal infections, postoperative wound infections, skin and soft-tissue infections, together with other bacteria [13, 19]. These infections may be caused by enzymes produced by Bacteroides, which damage intestinal tissue and disrupt the epithelial barrier [12]. What factors are crucial deciding whether the bacteria perform as a commensal or pathogen is not clear. Within the Bacteroides fragilis group are seven subgroups, which are given in figure 2.

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Figure 2. Phylogenetic tree of the members of the Bacteroides fragilis group. The tree is based on 16s rRNA genes and the bootstrap value is 500 [20].

Several isolates obtained from faecal samples were identified as members of this group, like Bacteroides dorei, B. thetaiotaomicron and B. distasonis (reclassified as Parabacteroides distasonis)[20]. These isolates are described below.

1.1.2. Bacteroides thetaiotaomicronThis Bacteroides species is a symbiotic and a dominant member in the intestine of mammals including humans [13]. As previous described, Bacteroides have a broad spectrum of sources they can use to generate nutrients [13]. Genome sequencing of B. thetaiotaomicron revealed 172 glycosylhydrolyses containing of enzymes involved in degrading host-derived products, polysaccharide binding proteins and sugar-specific transporters. This flexibility gives B. thetaiotaomicron an advantage in an environment which is continuously changing, as is the case in the human gut [15]. Another beneficial characteristic of B. thetaiotaomicron is that it has an anti-inflammatory effect in the gut, thereby increasing the level of tolerance in the gut [12]. B. thetaiotaomicron (and other members of B. fragilis group) can produce polysaccharides which activate CD4+T cell responses. Activation of CD4+T cells can lead to both pro and anti-inflammatory responses, depending on the type of stimulants they get from the environment [13]. Evidence has been found that Bacteroides are able to prevent abscess formation by acting in that way. However, the production of the polysaccharides by members of the B. fragilis group have also been associated with development of abscess [13]. B. thetaiotaomicron has also been isolated from abdominal infections, skin infections and bloodstream infections [19].

The description above clearly indicates that the presence of Bacteroides species in the gut influences the health status of the host in both positive and negative manners. Remarkably, is that less than 1% of the bacteria in the gut are of the B. fragilis group, while it is the most commonly isolated anaerobic pathogen [12, 13]. For this reason members of the B. fragilis including B. thetaiotaomicron group are worth investigating in this project.

1.1.3. Parabacteroides distastonisParabacteroides are like Bacteroides obligate anaerobic, non-spore forming and non-motile, Gram-negative rods [20]. Parabacteroides are members of the Bacteroides subgroup of the phylum

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Bacteroidetes and consist of two species: Parabacteroides distasonis and Parabacteroides merdae [13, 20]. As been mentioned above, people suffering from inflammatory and age-related diseases have an increased number of Parabacteroides in their gut and this might be an indication that these bacteria can be involved in pathogenicy. Furthermore, Parabacteroides species have been isolated from abdominal infections, abscesses and skin infections, while Parabacteroides merdae has also been isolated from diabetic foot infections [19].

1.1.4. Odoribacter splanchnicusOdoribacter belongs to the family of Porhyromonadaceae in the order Bacteroidales [21]. These are rod-shaped, Gram-negative, non-pigmented, non-spore forming and non-motile bacteria, which produce compounds which are smelly, as the name already suggested (odori)[21]. This is caused by its ability to produce H2 and H2S. The organism has some similarities compared to the genus Bacteroides, but the organism differs in biochemical characteristics and in its 16S rRNA genes [21]. As a commensal the organism plays a role in digesting food sources which are not digestible by the host. But Odoribacter has in the human gut the potential to become a pathogen as well [22]. For example, it can be involved in the formation of abdominal abscesses [21]. The organism has also been isolated from blood samples of a patient suffering from pelviperitonitis [21]. As this bacterium can be a potential pathogen, it might be interested to investigate.

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1.2. AnaerostipesAnaerostipes are a group of bacteria and it is estimated that this genus represents more than 2% of the total microbiota [23]. This species shows great similarity with Eubacterium and is belonging to the Lachnospiracae family [23]. These bacteria are strictly anaerobic, non-sporeforming and rod-shaped. In the exponential growth phase the cells are Gram positive, while in stationary phase the cells are Gram-negative [24]. Anaerostipes have the ability to produce butyrate from lactate and acetate and these endproducts have been associated with beneficial effects on the human health [1, 25].

Anaerostipes have not been associated with infections or clinical failures and has not been classified as a potential pathogen.

Figure 3. Phylogenetic tree from Anaerostipes species based on 16S rRNA sequences and the numbers in the figure are bootstrap values n=500.

The tree shows that Anaerostipes have some relationship with Clostridium species, as they both belong to the group of Lachnospiracae. Clostridium species in the human gut play a role in degradation of plant-based material, but have also been involved in infections [11, 26]. Infections involving Clostridium species led in 6% of the cases to clinical failure or to reinfection in 10% of the cases [27]. As Clostridium species might be a potential pathogen, this increases the relevance of investigating this species as well. The information available of Clostridium species is also used to investigate the Anaerostipes isolated during this project. The lack of information about Anaerostipes makes this an interesting species to investigate. Also despite the recent discovery of Anaerostipes the ARDB shows two different antibiotic resistance genes; one for tetracycline and one for bacitracin [28, 29]. This can be an indication that Anaerostipes might carry more resistant genes, which have not been indentified yet.

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1.3. AntibioticsAntibiotics are a drug or group of chemicals, which are naturally produced by fungi and microorganisms,to kill or inhibit growth of bacteria [30]. Antibiotics were originally discovered in fungi as a natural bacterial killer agent, but nowadays synthetic antibiotics are produced as well [30]. Antimicrobial agents can be described as bacteriostatic or as bactericidal [2, 31]. Bacteriostatic agents inhibit growth or multiplication of the bacteria [2, 31]. The immune system of the host gets in this way more time to clarify the bacteria on its own. Bactericidal agents kill the bacteria, which do not require a competent immune system of the host [2, 31]. Antimicrobial agents can act on different cell components. The main targets of antimicrobial agents are inhibition of cell wall synthesis, inhibition of ribosome function, cell membrane function and nucleic acid function, inhibition of metabolism [30].

All antibiotic compounds described below will be tested on the Bacteroides and Anaerostipes isolated from the faecal samples. Choice of these antibiotics is based on resistance found for these bacteria which is reported to the Antibiotic Resistance Genes Database (ARDB) and Clinical and Laboratort Standards Institute (CLSI) 2014 [32].

Classes of antibiotics

1.3.1. -lactamsΒ The β-lactam antibiotic family includes penicillins, cephalosporins, carbapenems, monobactams and β-lactam inhibitors [30]. The name β-lactams is based on the structure of the molecule as it consists of a β-lactam ring [30]. This ring is needed to inactivate transpeptidases needed for cell wall synthesis [30]. The nucleus of this ring appeared to be the key component, 6-aminopenicillanic acid [33]. Novel β-lactam agents were produced by adding and adapting the side chains to this key component.

Β-lactams are often used in combination with a β-lactamase inhibitor like clavulanate, sulbactam or tazobactam, which irreversibly inhibit β-lactamase enzymes. Β-lactam β-lactamase inhibitor combinations are effective in treating anaerobic infections and are therefore used. Examples of these combinatory therapies are amoxicillin-clavulanate and piperacillin-tazobactam agents.

1.3.2. -lactams : cephalosporinsΒAnother family of β-lactams discovered are the cephalosporins [33]. Several generations of cephalosporins have been synthesised which have a broad-spectrum antibiotic activity, in total four generations [33].

These different generations have different spectra of activity and timing of introduction. The first generation of cephalosporins have a good activity against Gram-positives [30]. The second generation cephalosporins have a better activity against Gram negatives and less activity against Gram positives compared to the first generation [30]. Examples of these second generation cephalosporins are cefoxitin and cefotetan. The third generation has a good activity against Gram-negative bacteria and variable activity against Gram positives, like for example cefotaxime and ceftazidime [30]. The fourth generation has a broad spectrum activity against Gram negatives and Gram positives [30]. The fourth generation cephalosporin is cefipime [33].

Bacteroides are Gram-negative anaerobes, and are expected to be susceptible for second, third and fourth generation cephalosporins. But anaerobes are known to produce β-lactamases, which possible

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led to resistance against cephalosporins [18, 34]. Cephalosporins were often used to treat intra-abdominal infections caused by B. fragilis, but resistance have been led to clinical failures and are subsequently no longer recommended for usage [18, 35]. For these reasons second and third generation cephalosporins and β-lactam antibiotics will be tested.

1.3.3. -lactams : carbapenemsΒCarbapenems are a group of antibiotics which is active against a wide variety of aerobic and anaerobic Gram positive and Gram negative bacteria, including the multiple resistant bacteria like Pseudomonas, Enterobacter, Actinobacter and enterococci [19, 35, 36]. Examples of carbapenems are imipenem, meropenem, doripenem and ertapenem. Carbapenems have been used to succesfully treat abdominal infections, meningitis, community-acquired and nosocomial pneunomia and neurtopenic fever [35].

Resistance genes for β-lactam have not been identified in Anaerostipes yet. But literature mentioned that anaerobic bacteria possess several natural mechanism that prevent the effect of β-lactams [18, 34]. These mechanisms include production of β-lactamases, production of low-affinity binding proteins and decreased permeability of the cell membrane[31, 33, 35, 37].

1.3.4. Chloramphenicol Chloramphenicol is an active against a broad spectrum of bacteria which includes Gram positive, Gram negatives and both aerobes and anaerobes [30]. It is a bacteriostatic agent and inhibit protein synthesis [35, 38]. Protein chain elongation is inhibited by binding to the bacterial 23S rRNA of the 50S ribosomal subunit [30, 35, 38]. As a consequence the peptidyl transferase is inhibited. Chloramphenicol has been used to treat anaerobic infections and infections of the central nervous system [35]. However, the agent has a risk of aplastic anaemia (toxicity), making this nowadays not the preferred drug of choice [35].

Resistance against this agent has been rare, but some Bacteroides species appear to be resistant against chloramphenicol, including clinical failures [35]. For this reason the efficacy of chloramphenicol against Bacteroides species will be tested.

Resistance in Anaerostipes for this agent are not known, but Clostridium species may be resistant for this agent. Three genes have been known called cata11, cata15 and cata16 [22, 30, 39, 40]. All of these genes encode for chloramphenicol transferases [40]. This enzymes destroys the activity of the agent by acetylation. These genes have been known to be present on transposable elements and have been known to transferred between different bacteria species as well, making it possible Anaerostipes possess this resistance as well [39, 40].

1.3.5. AminoglycosidesThe aminoglycosides are over half a century old and the first that has been discovered is streptomycin [30]. Aminoglycosides, like streptomycin were discovered in Streptomyces species, leading to the discovery of kanamycin and neomycin as well [30]. Aminoglycosides act by inhibiting the synthesis of the proteins involved in cell membranes and have in general a broad spectrum.

Anaerostipes do not have resistance genes for aminoglycosides. However, Clostridium species might possess a gene that induce resistance for streptomycin and ampicillin [41]. This gene is widespread among enterococci and staphylococcus species species as well [30, 41]. In Bacteroides several genes

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responsible for aminoglycoside resistance has been identified, such as anti6ia and aphii3a, but also erm genes might induce this resistance [38, 42, 43].

1.3.6. Macrolides-lincosamide-streptogramin B (MLS) antibioticsMacrolides are another class of antibiotics. Examples of macrolides are erythromycin, azithromycin and clarithromycin. Macrolides are active against Gram positive non-spore forming anaerobic bacilli and clostridia and are a bacteriostatic agent. The have a moderate effect on gram-negative bacteria. The compounds bind to the 50S subunit of the bacterial 70S rRNA complex and inhibits protein synthesis. Inhibiting protein synthesis subsequently inhibits cell replication and other processes needed for cellular growth [6, 19, 35].

Erm genes are responsible for resistance against MLS antibiotics and are widespread among a large group of bacteria including Bacteroides and Clostridium species. One gene called ermB is for example being identified in Acinetobacter, Bacillus, Bacteroides, Clostridium, Enterobacter, Eubacterium, Enterococcus, Klebsiella, Ruminococcus, Staphylococcus and Streptococcus [30, 44] [11]. As these genes can be widespread it is possible erm genes are present in Anaerostipes as well. For Bacteroides species there has even been an transposon carrying the ermB gene identified, making it highly likely that Bacteroides found in our project carry that gene as well [38].

1.3.7. TetracyclinesTetracyclines inhibit bacterial protein synthesis by binding to the ribosome [30, 45]. In this way the binding of amino acyl tRNA with the ribosome is prevented [28, 45]. This prevention is reversible, making the antibiotic a bacteriostatic agent [45]. Once tetracycline has been used to treat anaerobic infections [30, 45]. But development of resistance to virtually all types of anaerobes has reduced the efficacy of these drug [28, 46]. New synthetic tetracylines like doxycycline and minocycline remain more active compared to the original one [35, 46].

Tetracycline resistant genes has been found in both Bacteroides species and in Anaerostipes species [28, 45, 46]. These genes have been registered in the ARDB as well. Resistance for this antibiotic has been that common it is often not tested when performing susceptibility testing in a clinical setting [28, 45, 46]. Bacteroides and Anaerostipes will be tested on their ability to induce resistance against that agent as a confirmation.

1.3.8. Bacitracin Bacitracin is an antimicrobial produced by Bacillus. This agent consist of a mixture of high-molecular-weight polypeptides. This antibiotic causes disruption of the cell wall synthesis by binding to a pyrophosphate carrier in bacterial cytoplasmic membrane. Pyrophosphate is responsible for synthesis and transport of peptidoglycan subunits. Recycling of pyrophosphate units is important to enable further transport of subunits [47]. Bacitracin binds to pyrophosphate and thereby prevents recycling of this compound [47].

Bacitracin resistance has been found in Anaerostipes and this resistance has been registered in the ARDB as well [47]. For this reason resistance against bacitracin for Anaerostipes will be tested.

1.3.9. VancomycinVancomycin is a glycopeptide which inhibits cell wall synthesis [42]. Cell wall synthesis in Gram positives can roughly be divided in three steps. The first involves synthesis of cell wall precursors in the cytoplasm, the second step involves transport from the cytoplasm to the cell wall and transfer

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outside the membrane [42]. The third and final step involves attachment by a trans glycosylation reaction of the precursor to the cell wall [42]. Vancomycin is not able to penetrate the cell wall and must therefore act on the final cell wall synthesis step. Inhibition of this transglycosylation causes accumulation of cell wall precursors in the biosynthetic pathway and in the cytoplasm [42]. Cell wall synthesis in Gram positives are different compared to Gram negatives, causing to have no effect on the latter one [48, 49].

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1.4. Antibiotic treatments at intensive care: SOD vs SSDAntibiotic are administrered to treat and avoid infections. One group that is vulnerable for infections are patients on the intensive care (ICU). Two different antibiotic treatments are commonly known to be used on the intensive care, which are selective digestive tract decontamination (SDD) and Selective oropharyngeal decontamination (SOD).

SDD includes the administration of non-absorbable antibiotic is delivered straight into the digestive tract [50]. The aim is to prevent oropharyngeal carriage of potentially pathogenic microorganisms like Staphylococcus aureus, yeasts and aerobic Gram-negatives and consequently to avoid exogenous infections [51]. This treatment has first been used in the eighties and the effect of the treatment has been studied extensively [50]. The treatment includes administration of a variety of antibiotics like polymyxin E, tobramycin, amphotericin and third (cefotaxime) and fourth generation cephalosporins [51]. The drugs are chosen to minimise the disturbance of the anaerobic microbial flora in a patient [51]. This treatment shows to have several benefits in terms of: decreased mortality rate, lower airway and bloodstream infections, and decreased length of stay at ICU [50]. However, the treatment has also some drawbacks; potential for antibiotic resistance and modification of the microbiota, efficacy has primarily been seen in ICU’s with a low prevalence resistance and mostly in Europe, some studies suggest ICU in which MRSA was endemic SSD treatment leads to higher MRSA infections rates [50, 52].

With SOD the administration of antibiotic is done orally [50]. SOD contains the application of topical antibiotics in the oropharynx only, thereby omitting the intestinal components [51]. This treatment includes the same antibiotic used with SDD treatment, except no cephalosporins were used. This treatment has been postulated as an alternative to SDD for the prevention of ventilator associated pneumonia [50-52]. Comparable studies between the two different treatment conclude that both treatments decrease mortality in adults in intensive care [51]. However, it is not clear if and which of the two treatments is more effective [51].

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1.5. Effect of antibiotics on the microbial community in the gutAntibiotic treatment affects the composition of the microbiota of the host and also affects antibiotic resistance in gut [2]. During antibiotic administration the number of micro-organisms reduces and increases selective pressure in the gut and therefore increase the chance of transfer of antibiotic resistant genes [30, 31]. This effect is also illustrated in figure 4.

Figure 4. The effects of antibiotic treatment on the composition of microbiota. Adapted from Sommer, 2011. Before treatment different sensitivities for antibiotics and resistance occur in the gut. During exposure susceptible organisms will decrease in abundance, while resistant microbiota increase in microbiota, as they are not affected. After treatment the community will restore its abundance to previous levels, but some bacteria will be lost [2].

Studies have been done about the effect of the antibiotics on the composition of the gut (Dethlefsen et al., 2008). The treatment causes reductions in Firmicutes, but Bacteroidetes and Proteobacteria remained present in the gut [53, 54]. After treatment, the composition of the gut remains altered compared to the composition before the treatment. The recovery to a relatively stable microbial community took about one week, but treatment leads to complete loss of low abundance community members [54] [2].

In general the effect of antibiotic treatment can be divided into short-term induced and long-term induced antibiotic changes. Short term induced changes in the microbiota depends on the activity spectrum of the antibiotic; resistant or insensitive bacteria increase in abundance at expense of susceptible ones [2]. For example, eradication of Gram-positives after vancomycin administration creating opportunities for Gram-negative bacteria, which are not effected by this compound [48, 49]. Long term or repeated exposure of antibiotic leads to increased abundance of resistance species in the gut, caused by the growth advantage of resistant organisms during treatment, but also through exchange of resistant genetic elements between bacteria [2, 31].

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1.6. Mechanisms of antibiotic resistanceResistance of antibiotics of bacteria has become an increasing problem over the previous decades. The usage of antibiotics to tread infections in humans, but also in modern farming practice has led to increased resistance from potentially pathogen bacteria like methicillin-resistant Staphylococcus aureus, vancomycin resistant enterococci and pathogens like Klebsiella pneumoniae, Acinetobacter baumanii and Pseudomonas aeruginosa [1, 2, 30, 55, 56]. Consequently, treatment of these potential pathogens is getting more difficult.

Bacteria have developed a variety of resistance mechanisms which are described below. This resistance mechanisms depends on the which pathways are inhibited by the antimicrobial agents [2, 30, 57]. The main types of biochemical mechanisms developed by bacteria for defence against antibiotics are decreased uptake, enzymatic modification and degradation, efflux, altered target and overproduction of the target [37, 58]. Below is a picture (figure 5) of the different mechanisms of resistance developed over the previous decades, adapted from Byarugaba et al. 2010 [57].

Figure 5. Antibiotic resistance mechanisms developed by bacteria; A) efflux pump, B) transfer of antibiotic resistant genes by plasmids, C) Altering enzymes targeted by the antibiotics, D) development of enzymes that degrade the antibiotic. Each mechanism is described in more detail below.

1.6.1. Enzymatic modification and degradationAntibiotics often target enzymes involved in cellular processes. Bacteria overcome inactivation of these enzymes by modification of these enzymes. Modification can be done by blocking the target site for the antibiotics [2, 30, 37]. Another effective way bacteria use is production of counter-enzymes that degrade the antibiotics and thereby inactivating the antibiotic activity.

Enzyme modification to reduce affinity of antibiotics has been observed in Clostridium [36]. Vancomycin (Van) genes are present on the chromosome of the organism and allow peptidoglycan synthesis with a serine residue at the end of this molecule [36] [49]. This reduces the affinity for vancomycin [36].

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One example of enzyme degradation causing antibiotic resistance are b-lactamase enzymes. The β-lactams antibiotics are a group of antibiotics that contain a so called β-lactam ring [35]. This ring is important for the activity of these antibiotics and is needed to inactivate transpeptidases needed for cell wall synthesis [56, 57]. As a defence system against this antibiotic, bacteria have modified enzymes that act in different ways [57]. β-Lactamases hydrolyse β-lactams that contain ester and amide bonds, thereby inactivating the β-lactam. Not only β-lactams contain these molecular structures but also penicillins, cephalosporins, monobactams and carbapenems.

Hydrolysis or degradation of antibiotics can also be done by enzymes like esterases [37, 57]. Other enzymes that often undergo modifications to counteract antibiotics are aminoglycoside-modifying enzymes and chlorophenical acetyltransferases [36, 48]. Transferases are enzymes that bind adenylyl, phosphoryl or acetyl groups to the periphery of the antibiotic molecule [37, 57].

Most B. fragilis strains produce β-lactamases causing mainly resistance to cephalosporinases [35, 36, 48]. This cephalosporinase is chromosomal (cepA) and causes resistance to cephalosporins and aminopenicillins, but susceptibility to piperacillin and β-lactam/ β-lactamase inhibitor combinations remain [36, 48]. Other bacteria which have been found to produce β-lactamases are Clostridium, Porphyromonas and Fusobacterium [35, 36].

Not only β-lactams but also other antibiotics are degraded by enzymes produced in Bacteroides. Three genes which encode for enzymes that degrade tetracycline (Tet X1, tetX2 and tet32) have been identified in Bacteroides as well [35, 46]. TetX encodes for an enzyme that inactivate tetracycline in the presence of oxygen and is therefore an oxidoreductase [46]. Despite the requirement for oxygen this gene has been found in obligate anaerobes like Bacteroides [46].

1.6.2. Protein inhibition by antibiotic treatmentSome antibiotics interfere with proteins or with protein synthesis. In general protein synthesis can be blocked by binding to parts of the ribosomal machinery, which is a key organel involved in proper protein synthesis [6, 19, 35, 38, 45]. Blocking of the antibiotic activity can be done in different stages, for example, during the transcription via RNA polymerase, or by binding to the 50S ribosomal subunit, thereby blocking the binding of the antibiotic to the target [45, 59]. Another way to resist the antibiotics is by mutating the ribosomal subunit [45, 59]. Again this can block binding of the agent to the target site or the affinity of the target site can be decreased.

An example of protein modification leading to antibiotic resistance is the tetQ gene. This tetQ gene encodes for a ribosomal protection protein and is responsible for most tetracycline resistance observed in Bacteroides [46].

1.6.3. Blocking of DNA synthesisAnother target on antibiotics can be blocking of DNA synthesis. This synthesis required two different enzymes DNA gyrase and topoisomerase. Antibiotics like quinolones can bind to the DNA gyrase A subunit, preventing DNA synthesis [30]. However, mutations in the genes responsible for the two enzymes led to a reduced affinity to the DNA synthesis enzymes [2, 31]. Mutations occur at the DNA-binding surface of the enzymes and consequently the binding of the antibiotic is impeded.

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1.6.4. Efflux pumpsAccumulation of antibiotics in bacteria can be prevented by pumping the antibiotic out of the cell with membrane proteins. This is also called efflux pump, which is also illustrated in figure 6 [30]. The pumps can be specific to antibiotics and they can also transport multiple antibiotics of a wide range [47, 60, 61]. Examples of antibiotic targeted by efflux pumps are streptomycin, macrolide-lincosamide-streptogramin-B, quinolones and tetracycline [30].

The efflux pumps are probably the main mechanisms active in multidrug resistant bacteria. Although most of these efflux pumps are present in aerobes they are also present in Bacteroides fragilis as well. Homologs of MLS- resistance efflux proteins (mefA) and Bacteroides multidrug efflux (BME) pumps are described already [30, 35].

Figure 6. Mechanism of efflux pumps present in the bacterial cell membrane. Picture adapted from Giedraitiené, 2011 [37].

1.6.5. Reduced membrane permeabilityAnother way of preventing accumulation of antibiotics is by adapting the outer membrane permeability. In this way the drug uptake to a cell and transfer through the outer membrane in reduced [2, 31, 37]. Resistance to β-lactams in Bacteroides is not only established by production of β-lactamases, but resistance is also enhanced by reduced cell membrane permeability [36]. This is done by loss of porin in the membrane. This strategy is also found in bacteria that are resistant to quinolones [30].

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1.7. Mechanisms of genetic transferThere is evidence that antibiotic treatment increases the antibiotic resistance in a community. Furthermore, the selection pressure that occurs during antibiotic treatment led to an increased exchange of antibiotic resistance genes [1, 30, 31, 37]. Exchange of resistant genes can be done by different mechanism and different mobile genetic elements can be involved. First of all gene transfer can be done in two different ways: horizontally (bacteria:bacteria), (animal:animal) and vertically (mother: offspring) [1, 30, 31]. Gene transfer between bacteria occurs continuously, as this is the way for bacteria to adapt to environmental changes in an evolutionary way [1, 30, 31]. Besides the rapid adaptation bacteria can also introduce new traits [1, 30, 31]. Disadvantages are the spreading of antibiotic resistant genes and this creates potential for pathogens[1, 30, 31].

Gene transfer can occur with a variety of mobile genetic elements: these elements can be plasmid encoded, or on mobile pieces of chromosomal DNA [31, 58]. Three main mechanisms of gene transfer between bacteria are transformation, transduction or conjugation [31].

1.7.1. TransformationWith transformation bacteria are able to take up free DNA directly from the environment. During transformation DNA is taken up from the environment and incorporated into the host genome by homologous recombination or by transposition [30]. Bacteria able to do that are called ‘competent’ [30]. This ability to take up free DNA from the environment is rather limited. Microorganism like streptococci are only competent at a specific stage in their growth [30]. Some bacteria need specific requirements to be able to take up free DNA from the environment [30]. Due to the limits occurring for transformation, it is not expected that transformation is the main cause of the spread of antibiotic resistance genes [30, 31].

1.7.2. TransductionTransduction requires the presence of a vector, like a virus, phage or plasmid to transfer small pieces of DNA between bacterium [30, 31]. Sometimes bacteriophages does contain bacterial DNA instead of phage [30]. Bacteriophage harbour these genes from bacteria to be involved in important activities in the gut [1]. This bacterial DNA is packaged into the phage head and the DNA can be injected into bacteria [30]. Bacteriophages do not mediate horizontal gene transfer, but also play a role in forcing changes in the microbial composition [1]. Selective attack on dominant bacteria species in the gut creates new niches for less abundant species [1].

1.7.3. ConjugationConjugation is a method to transfer genetic material between one bacterium to another of a different mating type [30, 62].

Conjugation can be done with conjugative elements which contain all the genetic information required to transfer from one bacterium to another [30]. In general conjugative elements have a similar organization: conjugation, recombination, regulation and accessory modules [30]. Accessory modules often contain antibiotic resistant genes [30].

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DNA transfer between two viable cells can be done via conjugation, either with plasmids or with conjugative transposons [30, 31, 34, 63]. These transposons have characteristics of plasmids, transposons and phages [13, 62]. For example, conjugative transposons contain an excision and an integration mechanism similar as from plasmids and bacteriophages [13, 62].

Figure 7. Gene transfer between two bacterial cells (schematic). The integrated gene forms a circular DNA molecule (excision). The circular molecule is replicated into a nicked DNA molecule. At the same time is the conjugation apparatus formed (transfer). After transformation, in both bacterial cell is the circular DNA molecule replicated to create a fully circular plasmid molecule (replication). This molecule can be re-integrated into the chromosome of the bacterial cell (integration) or transferred to other bacterial cells (transfer).

The process of conjugation can roughly be divided into two steps. The first step requires formation of a relaxosome [6]. A DNA-protein complex, which will be transferred, is formed by factor encoded mobilization proteins present on the origin of transfer [6]. This will finally form a nicked DNA molecule. The second process required for transfer is formation of mating or conjugation apparatus; protein- based structure created between the donor and recipient cell membranes and facilitating the transfer of DNA [6]. This process is also summarized in figure 7.

1.7.4. Examples of gene transferEvidence is present that conjugation plays a role in the spread of antibiotic resistance genes. Anaerobic conjugative transposons often contain tetracycline resistance genes and are therefore called tet elements [6]. In both Gram positive and Gram negative bacteria are conjugative elements found that carry antibiotic resistant genes [6, 62]. One article (Stuart et al., 1992) investigates resistance in Gram-positive bacteria. Clinical isolates from Streptococcus suis carried resistant genes for tetracycline and erythromycin [63]. Identification and transfer of this conjugative element was

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possible between bacteria. Succesfull transfer led to resistance for both tetracycline and erythromcyin [63]. This article does demonstrate conjugative elements can spread not only single, but also multiple antibiotic resistance genes among a population. Transfer of multiple factors might be resulting in rapid rise in antibiotic resistance among the anaerobes.

Another example (Gupta et al., 2003) supports the hypothesis that gene transfer can occur between Gram-positive and Gram-negative bacteria. One conjugative transposon which carried an ermB gene has been found in several Gram positive bacteria, but has also been seen in several Bacteroides strains[38]. Conjugation of the gene between the different Bacteroides species was observed, but it was also observed the conjugative element was able to mobilize plasmids [38]. By analysing open reading frames from this plasmid, they found identical open reading frames from DNA segments originating from Clostridium perfringens strain [38]. This finding supports the idea that transfer of genetic elements can occur between Gram positive and Gram negative bacteria.

One study (Mazodier et al., 1989) shows that E.coli and Streptomyces species are capable to transfer genetic information with help of plasmids [64]. This studie shows transfer of genetic information between different bacterial species is possible. This enforces the theory that spread of antibiotic resistance is also possible between different bacterial species.

All these different studies support that plasmids containing transposons or conjugative elements can spread antibiotic resistant in a broad way. Plasmids can contain single and multiple antibiotic resistant genes and these genes can be transferred between different bacterial species with the same Gram-staining, between anaerobes and aerobes, and plasmids can also be transferred between Gram-positive and Gram-negative bacteria.

1.7.5. Mobile genetic elementsOther mobile genetic elements are not able to transfer between different host, but have the ability to move within the genome of the host [30]. Transposons and mobile introns are capable to translocate to new sites within the genome [30, 58].Insertion sequences are mobile elements that contain genes required for element mobility [30]. When insertion sequences contain accessory genes not involved in translocation they are called transposons [30]. Transposons are only able to move within the host, but can be transferred in mobile elements like plasmids of within conjugative elements [30]. More details about plasmids can be found in the section below.Integrons are genetic elements that have a site-specific recombination system that enables them to capture and mobilize genes [30]. These genes can include antibiotic resistant encoding elements. Integrons can be transferred between bacteria with help of plasmids or transposons [30].

1.7.6. PlasmidsA plasmid is a circular, mobile DNA molecule, which can act as vectors. Plasmids are DNA molecules which are able to replicate by themselves, as they can be considered as extra chromosomal elements that have their own origin of replication [30, 58, 62]. Plasmids are common present in bacteria, almost no bacteria genera without plasmids are known to exist [30]. Plasmids can contain genes encoding replication functions, but also genes encoding for replication and transfer [58, 65]. These genes allow plasmids to be transferred to new hosts via conjugation[62]. Other genes commonly found on plasmids are involved in generation resistance for antibiotics and for this reason plasmids

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are suspected to play an important role in the spread of antibiotic resistant genes [1, 31, 65]. If these resistant genes are present on a transposon this genes can translocate from the plasmid to the bacterial chromosome and be maintained in the absence of the plasmid [30].

Several articles have been published with evidence that plasmids carry antibiotic resistant genes and can spread these genes among a population [38, 46, 66, 67]. Several examples are described at the end of this chapter.

1.7.7. Gene transfer in the human gutThe gut is a perfect environment for gene transfer for several reasons. First, the gut contains a dense and diverse microbiota [6, 31]. Second, this microbiota is getting continuously in contact with new bacteria transported via incoming food. Third, biofilm which is present on the surface of food and on the mucus layer facilitate gene transfer between two bacterial cells [31]. For all these reasons there is the suspicion that the microbiota in the gut may serve as an reservoir of antibiotic resistant genes and therefore plays an important role in the spread and maintenance of those genes [2, 30, 31]. The suspicion is that especially transfer of plasmids plays an important role for the spread of resistance, which is also part of our hypothesis.

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1.8. Antibiotic resistance in anaerobes: BacteroidesBacteroides are often involved in mixed infections. These infections vary from intra-abdominal sepsis, gynecological infections, skin and soft tissue infections, endocarditis and bacteraemia [13]. Frequently prescribed antibiotics to treat these infections are β-lactams, carbapenems, clindamycin and metronidazoles [13].

Several studies have investigated the ability of Bacteroides to resist one or more antibiotics. For example, the tetracycline gene tetQ is present in Bacteroides. This gene is part of a rteA-rteB-tetQ operon, which is located on a mobile element CTnDot [13, 68]. Horizontal gene transfer increases the presence of tetQ gene in Bacteroides from 30% to 80% [13, 69]. Bacteroides species gain increasing resistance to cefoxitin, clindamycin, metronidazole, carbapenems, fluoroquinolones and β-lactam agents [13, 34, 38, 70]. The rates and type of resistance differ greatly per location and per Bacteroides strain. Treatment of Bacteroides infections can be done with the newer fluoroquinolones [13].

Also resistance to carbapenems remains to be rare, although cases have been described in which carbapenem resistance was induced due to of increased efflux pump activity [19, 71].

One article written by Wareham (2005), is about a Bacteroides strain, which is resistant to multiple antibiotics [72]. This strain has been isolated from a patient with sepsis. This strain was resistant to metronidazole, β-lactams and β-lactam/β-lactamase inhibitor combinations, carbapenems, macrolides and tetracyclines and only treatment with linezolid was sucessfull [72]. Treatment of linezolid (oxazolidinone antibiotic) was finally successful. This strain possessed many different antibiotic resistant genes; cfiA (β-lactam resistant gene), ermF (MLS resistant gene), tetQ2 and tetQ3 (tetracyline resistance), qyrA (quinolone resistant region), bmeB9 and bmeB15 (efflux pump genes) were all found in this single strain [72].

1.8.1. Antibiotic resistance in OdoribacterOdoribacter splanchnicus might posses a rsistant gene which induces low level resistance to variety of antibiotics in the classes of aminoglycosides and polymyxins [14, 21, 34, 35, 61]. This resistance is caused by a so called marC gene, which is a multiple antibiotic resistance (mar) gene [61, 66]. This chromosomally encoded gene encodes for an efflux pump [61].

1.8.2. Antibiotic resistance in ParabacteroidesParabacteroides seem to possess several antibiotic resistance genes as well; both Bacteroides and Parabacteroides possess β-lactamases that are able to degrade antibiotics like cephalosporins and penicillins [14, 19, 34, 73].

1.8.3. Antibiotic resistance in AnaerostipesAlso within the group of Anaerostipes antibiotic resistance occurs. According to the ARDB Anaerostipes can be resistant to tetracycline and to bacitracin [29, 47]. Members of the family are Anaerostipe hadrus, A. Butyraticus, A. Rhamnosivorans and A.caccae [23-25, 74]. The discovery of the species is quite recent and for this reason it might be possible Anaerostipes possess antibiotic resistant genes not identified before. This makes is also more difficult to compare our results with studies and results found in literature. Comparable studies might also be performed with studies

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done with Clostridium as this organism is known to possess antibiotic resistant genes and is closely related to Anaerostipes.

1.9. Research questions and approachIt is clear that antibiotic treatment has an adverse effect on the microbiota in the gut. It is not clear what happens to the commensal bacteria in the gut during antibiotic treatment and what happens to these bacteria in the gut after treatment has finished. Does antibiotic treatment induce resistance in the microbial community in the gut? If resistance is present in anaerobic isolates in the gut they might transfer this resistance to other bacteria present in the gut, including aerobes and potential pathogens. Are anaerobic bacteria in the gut a potential reservoir for antibiotic resistant genes?

This project is done to find information about the behaviour of microbiota in stress conditions. This project also contributes to the message that it is important to investigate anaerobic bacteria on resistance as well.

Faecal samples of ICU patients have been collected at different time points during their antibiotic treatment and after. Bacterial isolates from these samples will be characterized with different techniques.

The sensibility to antibiotics of the bacterial isolates will be tested, based on recommendations found in the ARDB and CLSI guidelines. If antibiotic resistance has been found, plasmids are tried to be isolated from the bacterial strains to perform transformation experiments.

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2. Materials and methods

2.1. Isolation of bacterial species from feacal samplesFrom two different patients were faecal samples taken at several time points. One patient has received SSD treatment (patient 120) and the other SOD treatment (patient I). A time scheme at which the different samples were taken can in found in figure 8. Faecal samples were taken: 1) at the start of the treatment, 2) during hospitalization and thus during antibiotic treatment, 3) and after hospitalization and thus after treatment was finished.

Figure 8. Time scheme (in days) used to collect faecal samples per patient treated with antibiotics. Patients 120 received SSD treatment and patient I SOD treatment. Samples were taken at the start of the treatment, during and after the treatment.

The faecal samples were cultivated with Reinforced Clostridial medium (RCA), Brain-heart Infusion medium (BM) or with Fastidous Anaerobe Agar medium (FAA), using the following antibiotics per plate: Tetracyclin (10 µg/ml), Ampicillin (100 µg/ml), cefotaxim (5 µg/ml) and imipenem (20 µg/ml). Based macroscopic and microscopic morphology and identification by 16S rRNA sequencing, A number of bacteria isolated per patient per media, identified and stored at -80 °C (table 1).

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Table 1. The table shows patient nr, time point (days), growth medium and antibiotics used to isolate the different Bacteroides strains. Abbreviations: RCA- reinforced clostridial media, FAA-fastidious anaerobe agar, BM- brain-heart infusion medium, T- tetracycline, C- cefotaxime, A-ampicillin, I-imipenem, B and FAA tetracycline and ampicillin.

Patient nr and time point (SSD)

Time point (days)

Medium Antibiotics Morpho-logy

Bacteria species

120 2 RCA T and C Bacteroides dorei120 8 FAA A and T Bacteroides dorei120 8 RCA T and C Bacteroides dorei120 14 BM I and A 1 Bacteroides dorei120 14 BM I and A 2 Bacteroides dorei120 14 FAA A and T 1 Bacteroides dorei120 14 FAA A and T 2 Bacteroides dorei120 14 RCA T and C Bacteroides dorei120 17 BM I and A Bacteroides dorei120 17 FAA A and T 1 Bacteroides dorei120 17 FAA A and T 2 Bacteroides thetaiotaomicron120 20 BM I and A 1 Bacteroides dorei120 20 BM I and A 2 Parabacteroides distasonis120 20 FAA A and T Bacteroides dorei120 20 RCA T and C Bacteroides dorei120 29 BM I and A Bacteroides dorei120 29 RCA T and C 2 Bacteroides dorei120 8 after FAA A and T 2 Bacteroides doreiPatient nr and time point (SOD)

Time point (days)

Medium Antibiotic Morphology

Bacteria species

I 2 B 2 Bacteroides thetaiotaomicronI 2 B 3 Odoribacter laneusI 2 FAA Bacteroides doreiI 10 B 1 Bacteroides sp. BLBE-2I 10 FAA 1 Candidatus Alistipes

marseilloanorexicusI 10 FAA 2 Bacteroides thetaiotaomicronI 10 B 2 Candidatus Alistipes

marseilloanorexicusI 10 B 3 Odoribacter laneus

2.1.1. DNA extraction and purificationDNA was extracted and purified for identification of the isolated bacterial strains from the patients feacal. DNA extraction and purification were performed with DNeasy Blood& Tissue Kit Qiagen isolation kit according to the manufactures instructions.

2.1.2. NanodropThe DNA purity and concentration was measured with the nanodrop. DNA concentrations were determined with the Nanodrop application Suite V4. Concentrations above 50 ng/µl are sufficient enough for continuing procedures and A260/A280 ratio above 1.8 is considered pure enough. Below 1.8 the DNA might be contaminated with protein and therefore, must be purified.

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2.2. Identification of patient samples by sequencing, blasting and building a phylogenetic tree

After isolating single colony cultures, the identity of the samples was verify by isolating the DNA as described above and performing 16S rRNA PCR. More details about the program and mastermix can be found in the Appendix. This PCR was done with two primers: Bac0027F and Uni1492R and the expected band size of 1500 bp was verified on an agaerose gel, before sending the sequences to GATC (www.GATC.com). After receiving the sequences the quality of these sequences were checked and adapted with Chromas version 1.45 and the corrected sequences were compared with the NCBI database by using BLAST.

2.2.1. Inferring phylogenetic relationships from sequence dataThe sequences were identified with known similar sequences in databases. The sequences from both the database and our own were used to build a phylogenetic tree. This tree can be used to identify the isolated strains. Sequences obtained were re-analysed with databases (BLAST) found in NCBI and RDP. Phylogenetic analysis was performed using the program MEGA6. The sequences were aligned pairwise and multiple wise with ClustalW program. Alignments were manually checked and when needed corrected. The tree was generated with maximum parsimony analysis [16]. To determine whether the data is supported, a bootstrap analysis was performed as well. The values are given in the figures which represents the phylogenetic trees. The main bacteria species identified were Bacteroides and Anaerostipes species.

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2.3. Physiological and biochemical characterisation of Bacteroides and Anaerostipes

2.3.1. Gram staining and microscopic examinationGram staining was done to characterize the different isolates. Gram stain was done by using the standard protocol and staining were directly examined under the microscope. A description of the technique can be found in the appendix.

2.3.2. Macroscopic examinationMacroscopic examination was the visual examination of bacterial growth on plates. This was done to verify pure cultures are growing with the same morphology and no contamination occurred. For Bacteroides growing of BM medium for 24/48 hours colonies were suspected to be circular, whitish, raised, convex and around 2 mm in diameter [75]. Anaerostipe strains growing on RCA medium are whitish or beige, circular, convex colonies with rough surfaces [25].

2.3.3. Motility testing Motility testing was done as part of the characterization of the isolates. Motility testing was done by taking bacterial samples directly and examine these samples under the microscope. A detailed description can be found in the appendix

2.3.4. Biochemical characterisation by API testBiochemical characterisation for Anaerostipes was analysed with API test CH50 (Bio-merieux, Marcy-l’Etoile, France) by following the manufacturer’s instructions. This test is done to see which sugars the bacteria are able to use in their metabolism pathways. Bacterial suspensions were distributed among wells, containing different sugar sources. Acidification of the sugar source turns the colour of the medium from purple into yellow and this indicates a positive result.

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2.4. Predict antibiotic resistance with ARDBAfter identification of the isolated strains the ARDB was used to find already known antibiotic resistant genes. The antibiotic resistance found in the database was used to make a list of antibiotics that can be tested on the identified strains.

2.4.1. Antibiotic resistant profile: AnaerostipesBelow is a table with an overview of Anaerostipes isolates. The ARDB was used to found if already known antibiotic resistance genes are identified in this species. Based on the genes found in the database and earlier growth conditions used a predicted antibiotic resistance profile is set up. This profile is given in table 2 below.

Table 2. Antibiotic resistance tested for the Anaerostipes isolates.Antibiotics tested Concentraction (µg/ml)Bacitracin 250Tetracyclin 10Ampicillin 100Imipenem 20Cefotaxime 5Streptomycin 25Chloramphenicol 12.5Erythromycin 100Vancomycin 10The antibiotic resistance database showed two known antibiotic resistance genes for Anaerostipes. One of the resistant genes is responsible for resistance against bacitracin and the other against tetracyclin. Furtermore, the bacteria were isolated with different antibiotics like ampicillin, imipenem and cefotaxime and these antibiotics will be tested as well. As Anaerostipes are also related to Clostridium this species was also researched with help of the database and based on that data will also streotomycin, chloramphenicol, erythromycin and vancomycin tested.

2.4.2. Antibiotic resistant profile: BacteroidesFor Bacteroides was also the ARDB used to find antibiotic resistant genes. An overview of these genes and antibiotics affected is given in the table below (table 3).

Table 3. Antibiotic resistance tested for Bacteroides isolates.Antibiotics tested Concentraction (µg/ml)Tetracyclin 10Ampicillin 100Imipenem 20Cefotaxime 5Streptomycin 25Chloramphenicol 12.5Erythromycin 100Kanamycin 50Cefoxitin 5The antibiotics will be tested depending whether the different isolates have the antibiotic resistance genes is common and whether the antibiotic is available or not. For example cefotaxime is only available in disc form and will therefore only be used in agar diffusion tests, while ampicillin and tetracyclin is available for both agar dilution (powder) and agar diffusion (disc) methods.

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Techniques to determine antibiotic resistance profile2.4.3. Agar dilution method

Agar dilution involves solid medium in Petri dishes containing a certain concentration of antibiotics. The concentrations at which the antibiotic is added are based on guidelines from CLSI and EUCAST at which a bacteria is still able to growth. Based on concentrations used and if growth occurs in the form of colonies the bacteria can be interpreted as susceptible or resistant.

2.4.4. Broth dilution methodWith the broth dilution test, antibiotics are directly added to liquid growth medium. Growth of the bacteria in the culture was checked visually and my microscopic examination.

2.4.5. Agar diffusion methodWith the agar diffusion method small commercially available discs containing antibiotic were placed on a non-selective medium, like Mueller Hinton or Wilkins-Chalgren medium. Before adding the discs onto the plates, bacteria were diluted until 0.5 mcFarland and spread on the entire plate with a sterile cotton swab in three different directions. The discs were put on top of the agar with help of tweezers. After 24 or 48 hours of incubation, diameters occur around the discs, also known as inhibition zones. Interpretation and size of the diameters occurring around the discs can be done with documents made by CLSI.

Table 4. Antibiotics tested for agar diffusion tests. These agar diffusion tests are divided in three different experiments: agar diffusion, double disk test and β-lactamase test. Antibiotic discs tested, each concentration of the antibiotic on the disc and the interpretation. S-susceptible, I-intermediate, R-resistant phenotype.

Anaerostipes (MIC values) Bacteroides (MIC values)

AntibioticsConc./ disc (µg) S (mm) I (mm) R (mm) S (mm) I (mm) R (mm)

Ampicillin 25 ≥ 14 < 14 ≥ 14 < 14Tetracyclin 30 ≥ 22 22-19 < 19 ≥ 22 22-19 < 19Imipenem 10 ≥ 22 22-16 < 16 ≥ 22 22-16 < 16Cefoxitin 10 ≥ 19 < 19 ≥ 19 < 19

2.4.6. -lactam disk testβΒ-lactams are a large group of antibiotics and bacteria have developed different mechanisms that induce resistant to these type of antibiotics. The β-lactam diffusion test is a method in which the disposition of the antibiotics allow to determine the resistant phenotype.

Table 5. Discs and antibiotics used to determine β-lactamase resistance in Bacteriodes. The interpretation: S-susceptible, I-intermediate, R-resistant phenotype.

Inhibition zones and MIC values

Ab disc diffusion testAbbreviation disc

Conc. (µg/disc)

S (mm) I (mm) R (mm)

Ceftazidine CAZ 30 ≥ 22 22-19 <19Amoxycillin /Clavulanic acid AMC 30 ≥ 19 19 < 19

Piperacillin /Tazobactam TZP 110 ≥ 20 20-17 < 17

Cefotaxime CTX 30 ≥ 20 20-17 < 17

Meropenem MEM 10 ≥ 22 22-16 <16

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Imipenem IPM 10 ≥ 22 22-16 < 16

Below the scheme showing in which order the disks were placed onto the plates (figure 9).

Figure 9. Scheme used for β-lactam disk test. The order of the antibiotics on the plate and concentration of the discs used are indicated. Also the different genes that can be determined with each antibiotic is given in the scheme. More details about the interpretation is given below.

Depending on the phenotype, shape and size of the inhibition zone the type of resistance can be determined. Interpretation is given below in the table (table 6).

Table 6. Phenotypes of the beta-lactamase test.Phenotype Resistance antibiotics -lactamaseΒCAZ <19 mm Ceftazidime(CAZ) resistance [56]. Extended β-lactamases (ESBL’s)

[56]AMC < 19 mmCTX <17 mm

Resistance to clavulanate (AMC) and synergy towards cefotaxime (CTX) [56].

ESBL’s: CTX-M types [56]

CAZ < 19 mmAMC< 19 mm

Resistance to clavulanate (AMC) and synergy towards ceftazidime (CAZ) [56].

ESBL’s: TEM/SHV types [56]

CAZ < 19 mmCTX<17 mmIPM> 22 mmMEM> 16 mm

Resistance to ceftazidime (CAZ), but susceptible for imipenem (IMP) [73].

AmpC phenotype [73]

MEM < 16 mmTZP< 17 mm

Resistance to meropenem (MEM) and synergy towards piperacillin/ tazobactam (TZP) [33, 70].

Carbapenems [33]

MEM< 16 mmIPM< 16 mmTZP <17 mm

Resistance to β-lactam-β-lactamase inhibitor combinations. Resistance to imipenem (IMP), meropenem (MEM) and piperacillin/ tazobactam (TZP) [70, 73].

Carbapenems: Metallo β-lactams (MBL’s) [33, 70]

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2.4.7. Double disk testThe double disc diffusion test was done to investigate the phenotype of erythromycin resistant bacterial strains. Two antibiotics (erythromycin and clindamycin) were tested and the distance between the two disks is 20 mm. The antibiotics tested and concentrations were given in table 7.

Table 7. Antibiotics tested during double disk test.Antibiotic tested (Oxoid) Conc./ disc

(µg)Abbreviation on disc

Erythromycin 15 EClindamycin 10 DAAfter 24/ 48 hours of incubation, three different phenotypes were expected; inducible, resistant and M-phenotype [76]. These phenotypes are also given schematically in figure 10.

Figure 10. Different possible phenotypes in case of erythromycin resistance. The phenotypes are named as Inducible, resistant and M-type [76].

Three different phenotypes are possible with this test. The inducible phenotype is characterized by a D-formed shape around the clindamycin disc, meaning the tested bacteria is resistant to both erythromycin and clindamycin [76]. The resistant phenotype shows no inhibition zones for around both discs, and this is interpretated the bacteria is resistant to both antibiotics [76].The third phenotype is also called the M-type. This phenotype shows a circular zone around the clindamycin disc, meaning the bacteria is resistant to erythromycin and suspectible for clindamycin [76].

2.4.8. E-testThe E-test can be performed to determine the (minimal inhibitory concentration) MIC value of an antibiotic on a certain bacterial strain. The test itself is a strip, containing a concentration gradient of the tested antibiotic. In this test an individual isolate was suspended in PBS and swabbed onto a MH/WC agar plate. The E-test is a plastic strip with an antibiotic concentration gradient, which is also indicated on the strip [35]. This strip was placed in the agar disk and an elliptical inhibition zone appears around the strip, which indicates the MIC [35]. The Etest has been performed to determine the MIC value of vancomycin for Anaerostipes isolates.

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A picture of this test can be found in figure 11.

Figure 11. Example of E-test. The picture shows a disc with five strips and around each stripe is a inhibition zone shown, indicating the MIC value for that antibiotic.

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2.5. Testing transformation of antibiotic resistance genesIsolated plasmids from Bacteroides and Anaerostipes were transformed into competent E.coli cells to determine whether transfer of genes between commensal bacteria and potential pathogens is possible.

2.5.1. Plasmid isolationPlasmid isolation was performed for bacteria that were able to grown with antibiotics in liquid growth medium (BM or RCA). Plasmid isolation was performed with the Thermo Scientific GeneJet Plasmid Miniprep Kit according to the manufactures instructions.

2.5.2. Preparing competent cellsFor the transformation experiments were compentent cell prepared. The details about the protocol can be found in the appendix. CCMB medium was used to prepare competent E.coli cells (JM109) and cells made were suitable for heat-shock transfection.

2.5.3. TransformationAfter isolation it is possible to directly transform the competent E.coli JM109 cells with the plasmid. This is done to simulate transfer and spread of antibiotic resistant genes between bacteria present in the gut. The antibiotic resistant gene present on the plasmid can be used to verify if the transformation of the plasmid was successful or not.

Transformation was also done to calculate the transformation efficiency rate. Calculating the transformation efficiency: number of colony forming units produced by transforming 1 µg of plasmid into a given volume of competent cells. Dilution rates of 10, 100, 1000 and 10000 times were made and spread onto LB plates with the proper antibiotic.

¿ colonies on plate

ngof DNA plated∗1000 (ngµg

)=Transformationefficiency ( transformant

µg)

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2.6. Agarose gel electrophoresisAgarose gels were prepared to visualize DNA. After 16s rRNA PCR a gel was prepared to verify if PCR went successful or not. After plasmid isolation, this DNA was also loaded to verify plasmid DNA was indeed isolated or not.

2.6.1. DNA gene-rulersFigure 12 below shows the three different DNA ladders used during gel electrophoresis.

Figure 12. GeneRuler DNA ladders used on agarose gels.

The following GeneRulers from Thermo scientific were used on the gels: GeneRuler 100 bp DNA ladder, GeneRuler 1 kb DNA ladder and GeneRuler 1kb plus DNA ladder.

2.7. PCR erm GenesIn order to identify which erythromycin resistant gene might be responsible for the resistant phenotype a PCR was performed. PCR was applied with specific primers for ermA, ermB and ermC genes. Details about the PCR programme and mastermix can be found in the appendix. The results of the PCR were examined on gel and positive results should give an expected band size of 645 bp for ermA, 639 bp for ermB and 642 bp for ermC.

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3. Results

3.1. Re-grown and obtain single coloniesThe purity of the strains was verified by Gram stain and by microscopic observations. Pictures of the Gram stain are shown in figure 13, one for every bacterial strain.

Figure 13. Gram stainings of the different bacterial strains. Bacterial strains identified were Anaerostipes rhamnosivorans 1y-2, Anaerostipes hadrus sp. 5.1.63FAA, Bacteroides dorei, Bacteroides thetaiotaomicron, Parabacteroides distasonis and Odori splanchnicus.

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The morphology of Anaerostipes are characterized by long rod-shaped cells (figure 13). Those cells can be alone, but can also group together. Bacteriodes dorei, Bacteroides thetaiotaomicron and Parabacteroides are also rod-shaped, although they are much shorter and smaller (figure 13). Odoribacter are described as fusiformed cells (figure 13).

After verifying the isolates were pure, the DNA from the bacteria was extracted, isolated and 16s rRNA PCR was performed. The products obtained from PCR were sent to sequencing. The sequences were analysed with Chromas and Blasted into the NCBI database. The results of the sequences of the different samples are shown in the tables 9 and table 10 below. The tables are organized by bacterial species.

Table 8. Isolates from patient 120 identified as Anaerostipes. Patients ID and the results from NCBI database are given.

NCBI database

ID sample Primers used sequencing

Name species Accession code Genbank

% Identity % Query

120B FAA AT A Bac0027F Anaerostipes rhamnosivorans sp. 1y-2 JX273468.1 99 100Uni-1492R

120C BM IA 2 Bac0027F Anaerostipes rhamnosivorans sp. 1y-2 JX273468.1 99 99Anaerostipes caccae sp. AIP 183.04 AY833660.1 97 99

120D FAA AT 1 Bac0027F Anaerostipes rhamnosivorans sp. 1y-2 JX273468.1 99 98Clostridium indolis AF028351.1 98 99

120E BM IA 1 Bac0027F Anaerostipes rhamnosivorans sp. 1y-2 JX273468.1 99 100Uni-1492R Anaerostipes hadrus 5.1.63FAA JF412658.1 97 100

120E FAA AT Bac0027F Anaerostipes rhamnosivorans sp. 1y-2 JX273468.1 99 98Clostridium indolis AF028351.1 97 100

120F RCA TC 2A

Bac0027F Anaerostipes hadrus sp. 5.1.63FAAJX273468.1

96 99

Anaerostipes hadrus NR_117138.1 96 99

120F RCA TC 2B

Bac0027F Anaerostipes rhamnosivorans sp. 1y-2JX273468.1

100 100

Uni-1492R Clostridium indolis AF028351.1 97 98

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Table 9. Isolates from patient 120 identified as Bacteroides, Parabacteroides and Odoribacter. The table gives the patients ID, DNA concentration, sequence length and the results from NCBI and RDP databases.

NCBI results

ID sample Name speciesAccession code Genbank

% Identity %Query

120C BM IA 2 Bacteroides dorei HS1 L3B CP008741.1 99 100

120D FAA AT 2 Bacteroides thetaiotaomicron 3443 AY895189.1 99 100

120E BM IA 2 Parabacteroides distasonis AB640686.1 99 100

NCBI results

ID sample Name speciesAccession code Genbank

% Identity %Query

IA FAA Bacteroides thetaiotaomicron JCM 5827 NR_112944.1 99 100

Bacteroides thetaiotaomicron VPI-5482 AE015928.1 99 100

IB 3B Odoribacter splanchnicus strain DSM 220712 NR_074535.1 99 99Isolates 120 C BM IA 2 and 120 E BM IA 2 has the same identity as obtained with the previous sequencing. This is not the case for 120 D FAA AT 2, which was identified as Bacteroides dorei in the previous identification, but now appear the be B. thetaiotaomicron. This had also been the case for IA FAA AT. IB B3 has in both cases been identified as Odoribacter.

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3.1.1. Re-identification of patient isolated sequences: Other BacteriaBesides identification of Anaerostipes and Bacteroides were also some other bacteria identified. These 16s rRNA sequences are given in the table below (table 11).

Table 10. The isolates and growth conditions and the identification done with NCBI and with RDP, showing the name of the species and accession number in the database.

NCBI results

ID sample Name species Accession code Genbank % Identity % Query

120B RCA TC Bacterium NLAE-zl-H380 JX006592.1 99 100Clostridium innocuum strain 607145/2009 HM008265.1 99 99

120C RCA TC Bacterium NLAE-zl-H380 JX006592.1 99 100Clostridium innocuum strain 607145/2009 HM008265.1 99 99

120E RCA TC Clostriduim innocuum strain 607145/2009 HM008265.1/HQ259734.1 99 100

NCBI results

ID sample Name species Accession code Genbank % Identity % Query

IA B2 E.coli LM997239.1 97 99

Mainly these bacteria were identified as Clostridium innocuum. Clostridium is Gram-variable, obligate anaerobic, rod-shaped bacteria [11]. These species are known the develop resistance against a variety of antibiotics. Clostridium strains were not taken into account, but will be used in another project from another colleague. Another bacteria isolated from the patient is E.coli. This bacterium is facultative anaerobe. The focus on this project is on obligate anaerobes. Therefore these bacteria will not be further used in this project.

In summary, for patient 120 are in total ten bacteria isolated from different time points at which faecal samples were collected. Seven of these isolates are Anaerostipes strains and three are Bacteroides, including one Parabacteroides. On patient I are two bacteria isolated, both are Bacteroides, including one Odoribacter.

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3.1.2. Phylogenetic tree building: AnaerostipesThe newly obtained sequences were used to build new phylogenetic trees. The same settings and programs were used to build these trees. The first tree shows the strains identified as Anaerostipes and the second tree shows the strains identified as Bacteroides (figure 14 and 15).

Figure 14. Phylogenetic tree of isolates identified as Anaerostipes.

Two groups can be seen in the tree; one group with the strains isolated from the patient and one group with sequences obtained from the NCBI database. The strains isolated from the patients are clustered around Anaerostipes rhamnosivorans, which can be expected based on the identification shown from the table above (figure 14 and table 9).

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3.1.3. Phylogenetic tree building: BacteroidesIsolates identified as Bacteroides, Parabacteroides and Odoribacter were clustered together and used to build a tree. This tree is given below in figure 15.

Figure 15. Phylogenetic tree of the patients isolates. These isolates were identified as Bacteroides, Parabacteroides or Odoribacter. Bootstrap values is 500.

This tree shows a illustrative way of the numbers given in table 10. The identities of the isolates given in this table are similar to the identities of which these isolates are clustered around. For example isolate 120 D FAA TC 2 and IA FAA were both identified as B. thetaiotaomicron in table 10 and in the tree these isolates are clustered around B. thetaiotaomicron as well (figure 15). The high bootstrap values indicate the data is strongly supported.

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3.2. API test AnaerostipesAnaerostipes were characterized with the API test CH50. The results are given in the table below (table 12). Pictures performed of the tests can be examined in the Appendix. The results of the API test were analysed after 48 hours of incubation. Isolate 120E BM IA 1 did not show growth and therefore no results were available.

Table 11. Results of the API test for Anaerostipes. Sugar acidification was considered as a positive result (+), no fermentation is considered as negative result (-). In some cases the colour of the sloths was light blue or green which is considered as not clear (?).

Sugars tested

120B FAA AT

120C FAA AT 2

120D FAA AT 1

120E FAA TC

120F RCA TC 2A

120F RCA TC 2B

Sugars tested

A.Rhamnosivorans [25]

Clostridium indolis [11]

Glycerol + - ? ? - ? Glycerol - +D-arabinose + ? + + - + D-arabinose ? +L-arabinose + - + + - + L-arabinose - +Galactose + + + + - + Galactose + -D-mannose + + + + - + D-mannose + -Rhamnose + + + + - + Rhamnose + -Cellobiose ? - - - - - Cellobiose - +Melibiose + + + + - + Melibiose - +Sucrose + + + + - + Sucrose - +Amidon/ starch

? - - - - - Amidon /starch

- +

Sugars tested 120B FAA AT

120C FAA AT 2

120 D FAA AT 1

120E FAA TC

120F RCA TC 2A

120F RCA TC 2B

Sugars tested

A. Rhamnosivorans [25]

A. Hadrus [23]

D-mannose + + + + - + D-mannose + -Rhamnose + + + + - + Rhamnose + -N-acetyl-glucosamide

+ + + + - + N-acetyl-glucosamide

+ -

Cellobiose ? - - - - - Cellobiose - +Sucrose + + + + - + Sucrose - +Melezitose ? - - - - - Melezitose - +

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The results of the API test were compared with reference strains from literature. The complete table can be examined in the Appendix. In the table above were only the differences between the reference strains compared to the isolates. Similar results between the reference strain and the isolates were indicated with a colour.

The results above have the highest similarities with A. rhamnosivorans (more than 50%) (table 12). Only 120B FAA AT has some similar sugar metabolism compared to Clostridium indolis. 120F RCA TC2A shows similar carbohydrate metabolism as A. hadrus. The sequence data for this isolate also suggest this species is A.hadrus.

The results of the API test were combined with the results of the Gram staining, sequences and tree building to identify the Anaerostipes species. Based on all these results most Anaerostipes isolates were identified as A. rhamnosivorans (table 13). The only isolates which the identity is not sure is 120F RCA TC 2A, this isolates is probably Anaerostipes hadrus sp. 5.1.63FAA.

Table 12. Identification Anaerostipes isolates from patients faecal.

Isolate ID Anaerostipes species120B FAA AT A Anaerostipe rhamnosivorans sp. 1y-2120C FAA AT 2 Anaerostipe rhamnosivorans sp. 1y-2120D FAA AT 1 Anaerostipe rhamnosivorans sp. 1y-2120E BM IA 1 Anaerostipe rhamnosivorans sp. 1y-2120E FAA AT Anaerostipe rhamnosivorans sp. 1y-2120F RCA TC 2 A Anaerostipes hadrus sp. 5.1.63FAA120F RCA TC 2 B Anaerostipe rhamnosivorans sp. 1y-2

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3.3. Antibiotic resistance profileThe antibiotic resistance profile of the isolates was tested by different methods based on the recommendations of the guidelines of EUCAST and CLSI 2013.

3.3.1. Agar dilution testThe table below shows an overview of the different antibiotics tested (table 13). The patients number and growing conditions are shown, the species, antibiotic tested and the results. The experiments were performed multiple time and abberant results were given in the table as well.

Table 13. Agar dilution test of the individual isolates. Growth of the culture was interpreted as resistant (R) and no growth was interpreted as sensible (S). Abbreviations used: BAC- bacitracin, TET-tetracycline, FOX-cefoxitin, CHL-chloramphenicol, ER-erythromycin, KAN-kanamycin, AMP-ampicillin, STR-streptomycin, VAN-vancomycin, IPM-impenem. N.T-not tested

Ab tested BAC TET FOX FOX CHL CHL ER KAN AMP STR VAN IPMConc.(µg/ml) 250 10 5 8 12.

550 10

0200 100 100 40 20

120B FAA AT A

Anaerostipes rhamnosivorans sp. 1y-2

R R N.T R R S R R R R S R

120C FAA AT 2

Anaerostipes rhamnosivorans sp. 1y-2

R R S R R S R R R R S R

120C BM IA 2

Bacteroides dorei HS1 L3B

N.T R R R S N.T S R R S N.T R

S

120D FAA AT 1

Anaerostipes rhamnosivorans sp. 1y-2

S R S R S S R R R R S R

120D FAA AT 2

Bacteroides thetaiotaomicron 3443

N.T R R R S N.T R R R R N.T S

S

120E BM IA 1

Anaerostipes rhamnosivorans sp. 1y-2

R R R R R S R R R R S R

120E FAA AT

Anaerostipes rhamnosivorans sp. 1y-2

S R R R S S R R R R R R

120E BM IA 2

Parabacteroides distasonis

N.T R S R S N.T S S R S N.T RS R

120F RCA TC 2 A

Anaerostipes hadrus sp. 5.1.63FAA

S R S R S S R R R R R R

120F RCA TC 2 B

Anaerostipes rhamnosivorans sp. 1y-2

S R R S S S R R R R S R

Ab tested BAC TET FOX FOX CHL CHL ER KAN AMP STR VAN IPMConc. (µg/ml) N.T 10 5 8 12.

5 N.T 10

0200 100 100 N.T 20

IA FAA 1 Bacteroides thetaiotaomicron 3443 N.T

S R R S N.T

R R R R N.T

R

IB3B Odoribacter N.T R R R S N.T R R R R N.T S

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splanchnicus S SAll Anaerostipes isolates were resistant to tetracycline, erythromycin, kanamycin, ampicillin, streptomycin and imipenem. All Bacteroides strains were resistant to cefoxitin, kanamycin, ampicillin and streptomycin. Both different bacterial groups were multiple resistant.

Anaerostipes: Bacitracin resistanceFor bacitracin was resistance found for Anaerostipes isolates,but this was only the case for the bacteria that were isolated in the earlier stages (8, 14 and 20 days) during antibiotic treatment. At the end of the treatment the Anaerostipes are suspectible for bacitracin (17, 20 and 29 days). It might be the Anaerostipes that were resistant to bacitracin, were not resistant for the antibiotics used for treatment and where therefore affected.

Anaerostipes: Cefoxitin resistanceThe results for cefoxitin are remarkable for some samples, like for example 120F RCA TC 2A. This isolate was susceptible for the lower concentration tested, but resistant for the higher concentration that was tested. This seemed also be the case for 120C FAA AT2 (A.rhamnosivorans) and for 120D FAA AT 1 (A. hadrus). However, the resistant MIC values obtained from EUCAST for cefoxitin are 64 µg/ml. Concentrations we tested are below that values explaining the resistance to a concentration of 8 µg/ml as well. The therapy that was given to patient 120 includes cephalosporins and administration of this antibiotic affects the composition of the microbiota. The disc diffusion was done for this antibiotic to confirm the effect of this antibiotic on the anaerobic bacteria.

Anaerostipes: Vancomycin resistanceFor vancomycin, this agent only affects Gram-positive bacteria and therefore was only tested on Anaerostipes. Two of the Anaerostipes strains were resistant to vancomycin. For the strains that were able to grow on vancomycin (120E FAA AT and 120F2 RCA TC 2A) the E-test was done to determine the MIC for vancomycin.

Anaerostipes: Chloramphenicol resistanceFor Anaerostipes, the chloramphenicol resistance is only induced in the first part of the antibiotic treatment and for the lower concentration tested (12,5 µg/ml). The MIC values from EUCAST explain why the chloramphenicol might have induced growth, as the intermediate value is 16 µg/ml and the resistant MIC is 8 µg/ml [32]. The higher concentrations tested did not led to growth for the Anaerostipes strains.

Bacteroides: Erythromycin resistanceFor Bacteroides only 120C BM IA 2 (B.dorei) and 120E BM IA 2 (Parabacteroides ) were sensible for both agents. The other strains tested were resistant to the used concentration.

Bacteroides: Streptomycin resistanceFor Bacteroides, resistance for streptomycin has been induced after 17 days of treatment for patient 120, suggesting antibiotic treatment has led to selection pressure of bacteria carrying the corresponding resistant genes.

Bacteroides: Imipenem resistanceResistance to imipenem was expected for 120C BM IA 2 (B.dorei) and 120E BM IA2 (Parabacteroides), based on the fact these bacteria where isolated on growth medium with this antibiotic. But also isolate IAFAA (B.thetaiotaomicron) seemed to be resistant for this antibiotic as well.

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Bacteroides: Tetracycline resistanceThe results for tetracycline resistance for Bacteroides strains were not consistent. Between the replicates some isolates showed different phenotypes. The phenotype for tetracyclin has been confirmed with the disc diffusion test.

Ampicillin resistance All isolates, Anaerostipes and Bacteroides were resistant for ampicillin. Ampicillin resistance was expected as this antibiotic was used for isolation of these strains from the patients faecal. However, not all Bacteroides strains were originally isolated with ampicillin. The disc diffusion test can be performed to find out the strains are truly resistant or not.

3.3.2. Agar diffusion testing of isolatesThe disc diffusion test was performed to confirm the results obtained from the agar dilution test (table 14). The disc diffusion test was performed multiple times, sometimes leading to different interpretative categories. With some the size of the zone fall within two different interpretative categories. In that case the interpretation is not determined (N.D).Table 14. The results of the disc diffusion test of each isolate. The diameter of the zones (in mm) and the corresponding interpretation (Resistant= R and sensible =S) is given as well.

Isolate ID species Ab disc test Ampicillin Tetracyclin Imipenem Cefoxitin120B FAA AT A

Anaerostipes rhamnosivorans sp. 1y-2

Diameter (mm) 22-44 12-15 14-41 16

Interpr. S R N.D R

120C FAA AT 2

Anaerostipes rhamnosivorans sp. 1y-2

Diameter (mm) 14-28 11-16 40 14-15

Interpr. S R S R

120C BM IA 2

Bacteroides dorei HS1 L3B

Diameter (mm) 0 12 38 18

Interpr. R R S R

120D FAA AT 1

Anaerostipes rhamnosivorans sp. 1y-2

Diameter (mm) 18-32 11-15 0-41 11-18

Interpr. S R N.D R

120D FAA AT 2

Bacteroides thetaiotaomicron 3443

Diameter (mm) 18 16 40 12

Interpr. S R S R

120E BM IA 1

Anaerostipes rhamnosivorans sp. 1y-2

Diameter (mm) 22-30 0-13 18-32 14-16

Interpr. S R N.D R

120E FAA AT

Anaerostipes rhamnosivorans sp. 1y-2

Diameter (mm) 18-41 12-13 08-28 14-15

Interpr. S R N.D R

120E BM IA 2

Parabacteroides distasonis

Diameter (mm) 33 23 38 22

Interpr. S S S S

120F RCA TC 2 A

Anaerostipes hadrus sp. 5.1.63FAA

Diameter (mm) 22-37 15-16 16-40 12-15

Interpr. S R N.D. R120F RCA TC 2 B

Anaerostipes rhamnosivorans sp. 1y-2

Diameter (mm) 22-42 14-16 16-38 14-16

Interpr. S R N.D R

Isolate ID species Ab disc test Ampicillin Tetracyclin Imipenem Cefoxitin

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IA FAA Bacteroides thetaiotaomicron 3443

Diameter (mm) 10 17 25 9Interpr. R R S R

IB3B Odoribacter splanchnicus strain DSM 220712

Diameter (mm) >40 >40 >40 >40

Interpr. S S S S

For the Anaerostipes, all individual isolates were resistant to tetracycline and to cefoxitin. All strains were susceptible to ampicilin as well. For the Bacteroides, B.dorei and B.thetaiotaomicron were resistant to tetracycline and cefoxitin. Just like the results obtained with the dilution test, the isolates were multiple resistant.

Anaerostipes: Imipenem resistanceFor imipenem the inhibition zones differ too much between the experiment to fall within one interpretative category. This phenomenom is seen with Anaerostipes isolates tested. For imipenem additional experiments are needed in the future.

Bacteroides: Tetracyclin resistanceResistance to tetracycline for Bacteroides strains was confirmed as well. Only 120E BM IA 2 (Parabacteroides) and IB3B (Odoribacter) were susceptible to tetracycline.

Bacteroides: Ampicillin resistanceThe disc diffusion test reveals that Bacteroides strains were resistant for ampicillin at the start of the treatment (day 14 for patient 120 and day 2 for patient I), but it is lost during the antibiotic treatment.

Bacteroides: Cefoxitin resistanceTwo strains were sensible for cefoxitin, which were Parabacteroides (120E BM IA 2) and for Odoribacter (IB3B).

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3.3.3. -lactamase test Β BacteroidesResistance to beta-lactam antibiotics has been demonstrated in the previous tests and the phenotype has been investigated with this experiment. The table below shows a summary of the results of the β-lactamase test and the phenotype(table 15 and figure 16). The β-lactamase test was performed multiple times and the diameter of the smallest and largest inhibition zone (mm) measured is shown as well.

Table 15. β-lactamase disc test of Bacteroides. The diameter of the inhibiotin zone and interpretative category is given. N.D= not determined. R-resistant, S- sensible.

Ab disc test

Isolate ID CAZ AMC TZP CTX MEM IPM Phenotype

120C BM IA 2 Diameter (mm)

0 0 26-30 0 0-34 37-42 ESBL’s: TEM and CTX-M

Interpr. R R S R N.D S

120D FAA AT 2 Diameter (mm)

0 8-12 20-36 0-12

0-28 0-46 ESBL’s: TEM and CTX-M

Interpr. R R S R N.D N.D

120E BM IA 2 Diameter (mm)

20-25 24-28

30-40 40 22-40 25-36 No beta-lactamases

Interpr. S S S S S S

Ab disc test

Isolate ID CAZ AMC TZP CTX MEM IPM Phenotype

IAFAA Diameter (mm)

0-8 0-9 16-20 0-9 9-28 24-25 ESBL’s: TEM and CTX-M

Interpr. R R R R N.D S

IB3B Diameter (mm)

>40 16 >40 >40 >40 >40 No beta-lactamases

Interpr. S R S S S S

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Figure 16. Pictures of the different Bacteroides isolates of which β-lactamase tests were performed. Abbreviations of the antibiotics used: piperacillin/tazobactam (TZP), imipenem (IPM), ceftazidine (CAZ), amoxycillin/clavulanic acid (AMC), ceofaxime (CTX), meropenem (MEM).

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Phenotype: ESBL’sIsolate 120C BM IA2 (B. dorei), 120D FAA AT 2 (B. thetaiotaomicron) and IA FAA (B. thetaiotaomicron) was resistant to ceftazidine. Ceftazidine can be used to detect ESBL’s. These isolated were furtermore resistant to cefotaxime and amoxicillin/clavulanic acid and this phenotype is caused by the possession of two beta-lactamses. These ESBL types are called TEM and CTX-M [33, 70, 73]. As expected the Bacteroides strains show resistance to beta-lactam antibiotics.

For 120E BMIA2 (Parabacteroides) and IB3B (Odoribacter) inhibition zones around each beta-lactamase tested were around 40 mm. This result indicates that these strains do not posses any mechanism or gene that induce resistance to beta-lactam antibiotics. These results are in line with results seen in the agar diffusion test for cefoxitin.

3.3.4. Double disk test: AnaerostipesResistant to erythromycin often induces resistance to clindamycin as well and this phenotype can be tested with the double disc test. The table below gives an overview of results of the double disk test (table 17). The inhibition zone (mm) was measured if this was visible and the interpretation is given below as well.

Table 16. Double disc test performed on Anaerostipes isolates. The inhibition zone measured, form of the zone and interpretation is given.Isolate ID Ab disc diffusion test Diameter measured (mm) Zone Interpretation120B FAA AT A Erythromycin 0 R

Clindamycin 22 O- zone M-phenotype120C FAA AT 2 Erythromycin 0 R

Clindamycin 25 O- zone M-phenotype120D FAA AT 1 Erythromycin 0 R

Clindamycin 21 O- zone M-phenotype120E BM IA 1 Erythromycin 0 R

Clindamycin 22 O- zone M-phenotype120E FAA AT Erythromycin 0 R

Clindamycin 25 O- zone M-phenotype120F RCA TC 2 A Erythromycin 0 R

Clindamycin 23 O- zone M-phenotype120F RCA TC 2 B Erythromycin 0 R

Clindamycin 21 O- zone M-phenotypeAs can be seen in table above, all Anaerostipes isolates were resistant against erythromycin (table 17). The M-phenotype induces resistance to erythromycin only and not to clindamycin.

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Figure 17. Double disc showing the M-phenotype of isolate 120D FAA AT 1 (Anaerostipes rhamnosivorans). Abbreviations: clindamycin (DA), erythromycin (E).

3.3.5. E-test: AnaerostipesAs two Anaerostipes strains (120E FAA AT and 120 F RCA TC 2A) were resistant to vancomycin during the dilution test, in which a concentration of 40 µg/ml was tested, an E-test with this antibiotic was done. The results were given in the table below (table 17).

Table 17. E-test performed with Anaerostipes resistant to vancomycin. The right column shows at which concentration the isolates were inhibited.

ID isolate Vancomycin (µg/ml)120E FAA AT 0.094120F RCA TC2 A 0.094For both tests the elliptic zone started at a concentration of 0.094 µg/ml. To determine wheter these isolates are resistant or sensible for vancomycin the MIC values were looked up; which is 2 µg/ml. The two strains can be tolerant to this agent untill a concentration of 0.094 µg/ml. Tolerance means that the two strains can resist the killing effect, but the cell growth is inhibited [77].

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3.4. Transformation experimentsSeveral isolates were resistance to antibiotic in Anaerostipes as well as Bacteroides. Because the resistanct genes can be lcoated in mobile elements like plasmids, we decide to performed plasmid isolation and transformation experiments. A total of 8 plasmids of Bacteroides and Anaerostipes strains were tried to be isolated and transferred into competent E.coli (JM109) cells.

3.4.1. Plasmid isolation Bacteroides and AnaerostipesPlasmids were isolated from bacteria that were able to grow in liquid medium containing antibiotics. The table below gives an overview which isolates where able to grow on which antibiotics in the indicated concentration (table 18).

Table 18. Plasmids isolated from the isolates able to grow on antibiotics. Antibiotics used were ampicillin (A), erythromycin (E), kanamycin (K) and tetracyclin (T).

Replicate Obtained from Bacteroides/ Anaerostipes

Plasmids Strains AbConc. Ab (µg/ml)

conc. Plasmids (ng/µl)

IAFAA Bacteroides thetaiotaomicron A 100 1142IAFAA Bacteroides thetaiotaomicron K 50 317

120B FAA ATAnaerostipes sp. 1y-2 rhamnosivorans T 10 353.5

120C FAA AT 2Anaerostipes sp. 1y-2 rhamnosivorans A 100 946

120C BMIA 2 Bacteroides dorei K 50 107518-sep 120C BMIA 2 Bacteroides dorei A 100 85.7

3-okt 120C BMIA 2 Bacteroides dorei A 100 493.6

120C BMIA 2 Bacteroides dorei E 100 1050IB3B odoribacter splanchnicus E 100 202.7

The two plasmids isolated from 120C BMIA 2 with ampicillin are basically replicates. Plasmids were checked on an agarose gel to verify plasmid DNA instead of chromosomal DNA was isolated. The results of the gel were given in figure 18 below.

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Figure 18. Plasmids isolated from Bacteroides and Anaerostipes. The isolate ID and antibiotics were given as well. Order of the gel; lane 1: 1 kb plus marker, lane 2: IAFAA (B.thetaiotaomicron) + ampicillin, lane 3: IAFAA (B.thetaiotaomicron)+ kanamycin, lane 4: 120B FAA AT (A. rhamnosivorans)+tetracycline, lane 5: 120C FAA AT 2 (A. rhamnosivorans)+ ampicillin, lane 6: 120C BMIA 2 (B.dorei)+ kanamycin, lane 7: 120C BMIA 2 (B.dorei)+ ampicillin, lane 8: 120C BMIA 2 (B.dorei)+ampicillin, lane 9: 1kb marker, lane 10: 120C BMIA 2 (B.dorei)+ erythromycin, lane 11: IB3B (Odoribacter)+erythromycin.

In total 8 plasmids were isolated, of which 3 were from patient I and the other 5 were from patient 120. The bands of the linear plasmids were not always clear, but for the visible bands can be said that all plasmids had a size above or around 10.000 bp.

3.4.2. Transformation in E.coli and comparison original plasmidTo test whether the plasmids isolated from Bacteroides and Anaerostipes are transferable, a transformation experiment has been performed. The plasmids were transformed into competent E.coli (JM109)cells and incubated in medium containing the associated antibiotics.

In case of growth on the medium the plasmids were isolated from E.coli and compared with the original plasmid on an agarose gel. Additionally an antibiotic resistance profile is made from E.coli.

Only two of the transformations were successful, which were isolates 120C BM IA 2 +erythromycin (13-10), 120C BM IA 2 +erythromycin (7-10) and IB3B+ erythromycin, corresponding to B.dorei and to Odoribacter (figure 19).

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Figure 19. Agarose gel of the original plasmids from Bacteroides and the plasmids transferred into E.coli. Order of the gel from left to right; Lane 1: 100 bp plus marker, lane 2 and lane 3: IB3B (Odoribacter) plasmids originated from Bacteroides, Lane 4: IB3B (Odoribacter) transformed plasmid E.coli, Lane 5 and lane 6: 120C BMIA2 (B. dorei) plasmids originated from Bacteroides, lane 7: 120C BMIA 2 (B.dorei) transformed plasmid E.coli, lane 8: 1kb marker.

The first two lanes show the bands that were linear plasmids from IB3B which originated from Odoribacter. The third lane shows the same plasmid transformed into E.coli which was isolated again. All lanes show one band which are around the same size (> 10.000 kb), suggesting that the plasmid originated from Odoribacter is successfully transformed into E.coli.

The righter part of the gel (lane 5-8) shows the original plasmid 120C BMIA 2 isolated from Bacteroides dorei. Lane 5 and 6 show plasmids originated from Bacteroides, and lane 7 shows the plasmid which was successfully transformed into E.coli. Again the bands are similar in size, suggesting the same plasmid was isolated in both Bacteroides and E.coli.

Based on the results in figure 19, the plasmid transformation into E.coli was successful. To test this plasmid is functional in E.coli, an antibiotic resistant profile was done as well.

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3.4.3. Antibiotic profiling of E.coliIn case E.coli cells grow in medium with antibiotics an antibiotic resistant profile was done to verify whether antibiotic resistant genes are present on the inserted plasmid or not and whether the plasmid is functional into E.coli. An overview of the antibiotics tested, concentrations and the result is given below in table 20.

Table 19. Antibiotic resistant profile of E.coli. The antibiotics tested per isolate are given, the results and the interpretation. Results are resistant (R) resulting in growth, or sensible (S) resulting in no growth.

Sample ID NCBI resultsAb tested

Conc. (µg/ml) Result Interpretation

120C BM IA 2

Bacteroides dorei HS1 L3B

erythromycin100

RGene on plasmid

ampicillin 100 S No genetetracyclin 10 S Gene on chromosomekanamycin 50 S Gene on chromosomechloramphenicol 50 S No gene

Sample ID NCBI resultsAb tested

Conc. (ug/ml) Result Interpretation

IB3B

Odoribacter splanchnicus strain DSM 220712

erythromycin100

RGene on plasmid

ampicillin 100 S No genetetracyclin 10 S No genekanamycin 50 S Gene on chromosomechloramphenicol 50 S No gene

For both E.coli containing the different plasmids, the same antibiotic were tested and for both plasmids the same results appear. The plasmids were isolated under the presence of erythromycin and the transformed E.coli is therefore also resistant to erythromycin. The transformation was successful and E.coli was able to express the genes on these plasmids.

For the other antibiotics tested, no further genes were present on the plasmid. Compared with the previous results susceptibility to ampicillin and chloramphenicol was expected as no resistance was found. Resistance was induced for tetracycline and kanamycin for isolated 120C BM IA2 (B.dorei) in previous results. Susceptibility to tetracyclin and kanamycin in E.coli suggests the responsible genes are present on the chromosomal DNA, or on other mobile elements.

Isolate IB3B (Odoribacter) showed resistance to kanamycin only and not for tetracyclin in previous experiments. Resistance for tetracyclin is not present in Odoribacter and resistance to kanamycin is caused by a gene which is present on the chromosome, or on other mobile elements.

3.4.4. PCR of Erm genesThe transformants that showed resistance to erythromycin, were tested for the presence for erm genes. A PCR for three specific erm genes was performed; ermA, ermB and ermC. The gel showed no bands for the tested transformants and therefore none of these genes were present in the

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transformants. The usage of controls gave a positive result for ermB only, the positive control for ermC was negative and for ermA was no positive control available.

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4. Final discussion and conclusionThe purpose of this project was to investigate whether antibiotic treatment affects the anaerobic flora in ICU hospitalized patients, by performing antibiotic resistant profiles on these anaerobic bacteria. In case antibiotic resistance was found the transferability of these genes were tested with transformation experiments.

4.1. Patients theraphyIn summary, two different patients were investigated during their antibiotic treatments. Patient 120 received SDD and patient I received SOD treatments. Both treatments have as a purpose to avoid aerobic infections, but to minimise the disturbance of the anaerobic patient’s flora at the same time. From the feacal samples taken during and after the treatment were bacteria isolated. From patient 120 were two different bacterial species characterised, which were Anaerostipes and Bacteroides. For patient I was mainly Bacteroides species isolated. The figure below shows which bacteria were isolated from which time points (figure 20).

Figure 20. Bacterial species isolated from each time point per patients.

For some of the feacal samples taken, no Anaerostipes or Bacteroides bacterial strains were isolated. This can be considered as a disadvantage. To make a better comparison, bacteria should have isolated from every time point available. Furtermore, the isolates used during this project only give a view of the microbial composition during the theraphy and not before of after the treatment.

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4.2. Effect of the theraphyDuring SSD treatment the patient receives different antibiotics; polymyxin E, tobramycin, cefotaxime and amphotericin [51, 52]. SOD treatment includes the same antibiotics except no cefotaxime is used during treatment.

Polymyxin E and tobramycin act on Gram negative, aerobic bacteria and are not adsorable in the gut, and have therefore no effect on the anaerobic bacteria in the gut [51, 52]. Amphotericin targets fungi and no bacteria and has no effect on the anaerobic bacteria in the gut either. However, cefotaxime is a third generation cephalosporin and has an effect on Gram-negative, Gram-positive bacteria including anaerobes and including Bacteroides species [33, 70].

In theory the cephalosporins used during antibiotic treatment would affect the composition of the microbial gut, for patient 120. However, cefotaxime has not been tested, instead cefoxitin has been tested. This second generation cephalosporin affects anaerobic bacteria as well, but this drug also induces beta-lactam activity [33]. To make a better comparison of the results, an overview of the results of the susceptibility tests done for both bacterial strains is given in table 20.

The table shows that most Anaerostipes and Bacteroides isolates are resistant to cefoxitin, indicating beta-lactam activity is present. This resistance also causes that SDD treatment does not influence the Anaerostipes and Bacteroides strains. But Parabacteroides is not resistant to this agent and might therefore be affected by SDD treatment. The number of Parabacteroides identified in the fecal samples are also much lower, compared to number of Bacteroides or Anaerostipes, indicating the number of Parabacteroides in the gut reduces as well. The role of Parabacteroides in the gut can be both commensal and pathogenic. Parabacteroides has been isolated from a number of infectious wounds, but one article (Kverka et al. 2012) also mentioned Parabacteroides reduces severity of intestinal inflammation in mice models [19, 78]. This suggests that the reduction of Parabacteroides numbers in the microbial gut, due to antibiotic treatment influences the functioning of the gut in a negative way and increase the inflammatory state of the gut.

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Table 20. Summary of the results of the susceptibility tests done of all the isolates. Abbreviations used: BAC- bacitracin, TET-tetracycline, FOX-cefoxitin, CHL-chloramphenicol, ER-erythromycin, KAN-kanamycin, AMP-ampicillin, STR-streptomycin, VAN-vancomycin, IPM-impenem.N.T-not tested

Ab tested BAC TET FOX IPM AMP KAN STR CHL ERY CA VAN

isolate IDConc. (µg/ml)

250 30 10 10 25 200 100 50 100 10 40

120B FAA AT A

Anaerostipes rhamnosivoran sp. 1y-2

R R R N.T. S R R S R S S

120C FAA AT 2

Anaerostipes rhamnosivoran sp. 1y-2

R R R S S R R S R S S

120C BM IA 2

Bacteroides dorei HS1 L3B

N.T R R S R R S S S N.T N.T

120D FAA AT 1

Anaerostipes rhamnosivoran sp. 1y-2

S R R N.T S R R S R S S

120D FAA AT 2

Bacteroides thetaiotaomicron 3443

N.T R R S S R R S R N.T N.T

120E BM IA 1

Anaerostipes rhamnosivoran sp. 1y-2

R R R N.T S R R S R S S

120E FAA AT

Anaerostipes rhamnosivoran sp. 1y-2

S R R N.T S R R S R S T

120E BM IA 2

Parabacteroides distasonis

N.T S S S S S S S S N.T N.T

120F RCA TC 2 A

Anaerostipes hadrus sp. 5.1.63FAA

S R R N.T S R R S R S T

120F RCA TC 2 B

Anaerostipes rhamnosivoran sp. 1y-2

S R R N.T S R R S R S S

Ab tested BAC TET FOX IPM AMP KAN STR CHL ERY

isolate IDConc. (µg/ml)

N.T 30 10 10 25 200 100 50 100

IA FAABacteroides thetaiotaomicron JCM 5827

N.T R R S R R R S R

IB3B

Odoribacter splanchnicus strain DSM 220712

N.T S S S S R R S R

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4.3. Susceptibility tests usedDifferent antibiotic assays have been used to investigate the effect of antibiotic treatment; agar dilution, agar diffusion and the E-test. The agar dilution test was done as a reference method. But not all results were consistent when performing this test. For example, cefoxitin was the result often sensitive with a concentration of 5 µg/ml, while a concentration of 8 µg/ml gave a resistant phenotype. The reason could be that antibiotics need to be added to the agar when the temperature of this agar is around 40°C to avoid degradation of the active compound. However, when the agar has been cooled down to much it becomes solid. Another possibility explaining the inconsistent result would be an unequal distribution of the antibiotic through the agar medium.

To confirm the results from the agar dilution test, the disc diffusion test has been performed. For tetracycline, ampicllin and cefoxitin the results were confirmed, but not for imipenem. In diverse literature sources the issue of developing trusted and standard methods for susceptibility testing for anaerobes has been mentioned. It was mentioned that anaerobic susceptibility testing has been more trouble shooting, due to insufficient or varied growth rates of anaerobes [35, 48, 79]. Other problems were that the intermediate range was too broad to be useful for determining resistance or susceptibility, results were not reproducible between different institutes and there was no correlation with the reference method (agar dilution method) [35, 48, 79].

4.4. Possible responsible resistant genes found in ARDB The section below discusses the resistance found during susceptibility testing and also dicusses the possible responsible genes which have been identified in the isolates before.

4.4.1. Resistance for beta-lactam antibioticsFor both Anaerostipes and Bacteroides species, resistance to cefoxitin has been found. Only Parabacteroides and Odoribacter were sensible for this agents. For Anaerostipes no beta-lactam resistance has been identified previously, but for Bacteroides several articles mention genes responsible for beta-lactam resistance.

Beta-lactam genes among BacteroidesFor example, the article of Dubreuil (Dubreuil et al. 2010) mentions that wild type strains of the B. fragilis group produce a chromosomal cephalosporinase called cepA [36]. These strains are resistant for cephalosporins and for aminopenicillins, like ampicillin and cefoxitin [48]. Isolate (120C BM IA2) B.dorei might possess this chromosomal gene, as this strain is resistant for previous mentioned antibiotics.

However, cefoxitin resistance Bacteroides thetaiotaomicron cannot be explained by the explanation above, as no resistance to ampicillin has been seen. But, the article mentions another mechanism which causes resistance to beta-lactam antibiotics, which is about loss of porin [36]. This mechanism has been described in Bacteroides and Parabacteroides distasonis [36]. In our case this resistant mechanism is only suitable for Bacteroides.

Resistance for cefoxitin might also be caused by the bl2e cfxa gene,which is present in all wild-type Bacteroides fragilis members [34, 56]. This gene encodes for a β-lactamase and causes resistance for cephalosporins [34]. Besides resistance to ampicillin this gene also can give resistance for tetracycline, clindamycin, piperacillin and cefoxitin [34]. This gene might also be in present in Bacteroides strains in both patients.

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Another reason to believe Bacteroides posses the bl2e cfxa gene are the results from the beta-lactasme test. The phenotype of the Bacteroides tested were are the same. Bacteria who are resistant to ceftazidime possess ESBL’s [34, 56, 73]. This phenotype is in line with the resistant induced by the bl2e cfxa gene found in the ARDB.

Concluding, several resistance mechanisms responsible for beta-lactams has been well described for Bacteriodes strains and explain the resistance found in our Bacteroides strains as well.

Beta-lactam genes among AnaerostipesFor Anaerostipes no genes responsible for beta-lactam antibiotics have been described. But many anaerobic bacteria, like Bacteroides and Clostridium are known to naturally produce beta-lactamases [3, 30]. Another possibility is that Anaerostipes strains posses a mechanims similar as described above for Bacteroides strains [6, 31].

4.4.2. Imipenem resistance The results obtained for agar dilution for imipenem has been inconsistent and the result for the β-lactamase test has been interpreted as susceptible in all Bacteroides and all Anaerostipes isolates from both patients. In literature, resistance for imipenem has been described rarely. Imipenem belongs together with meropenem to the carbapenems and have excellent activity against Bacteroides, including Bacteroides that produces β-lactamases [35]. Activity has also been towards multiple-resistant species like Pseudomonas, Enterobacter, Acetinobacter and enterococci [35]. Despite resistance is not common, one article (Nagy et al. 2011) mentions about 1% of the B.fragilis group has shown resistance towards imipenem, originating from countries in and around Europe including the Netherlands [19, 48]. Taken our results together with the rare resistance rates found for imipenem so far, we conclude that all isolates in patient 120 and patient I were susceptible to this antibiotic.

4.4.3. Tetracycline resistanceTetracyclin resistance among BacteroidesAll Bacteroides strains were resistant (B.dorei and B.thetaiotaomicron) to tetracycline as well. No resistant genes for tetracycline has been known for isolate 120E BMIA 2 (Parabacteroides) and for Odoribacter and therefore susceptibility for this antibiotic was expected, which was also the case.

Resistance for tetracycline has become that common in Bacteriodes clinical isolates, these strains are often not tested for susceptibility to this antibiotic [80]. Since the 1970’s resistant phenotypes for tetracycline has increased dramatically. TetQ is a widespread gene among Bacteroides strains and encodes for ribosomal protection resistance [80]. Detection of the tetQ gene among the different Bacteroides showed a high nucleotide identity (>80%) suggestion horizontal gene transfer has been responsible for the spread of this gene [80]. Evidence for horizontal gene transfer of the tetQ gene has been found in more articles. The tetQ gene has been found on conjugative transposons, of the socalled CTnDOT group and this transposon is present in Bacteroides strains [46, 80]. Sequence similarities between the different genes where around 96-100% and this finding suggest the spread of this gene has been through horizontal gene transfer [80]. Such high similarity sequences outrule the possibility of independent evolution of two versions of the same gene [80]. CTnDOT type elements can transfer antibiotic resistance if the donor is stimulated with low levels of tetracyclin[68]. This does not only led to maintenance of the resistant genes but also to spread of the tetQ and other genes present on CTnDOT type elements [80]. Low-level stimulation with tetracyclin

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might be induced continuously as the level of tetracyclin since it commercial usage has been increased 10.000 fold in fresh water [45, 46, 80].

Taken the widespread distribution of the tetQ gene among clinical isolates of Bacteroides species, it is highly likely this gene is also present in the gut of patient 120 and patient I.

Tetracyclin resistance among AnaerostipesTetracycline resistance for Anaerostipes has been found in all individual isolates. One gene found in the ARDB which might be responsible for this resistance is called TetO. This gene has been identified in Anaerostipes caccae only, but it is possible the gene might be present in A.rhamnosivorans and A. hadrus as well. Reasons to believe the gene might be present these strains is because this gene has been identified in varies organisms [29]. TetO was originally isolated from Streptococcus and in the article and evidence was found that the resistance gene originated from a Gram-positive bacterial strain [29]. TetO encodes for a ribosomal protection protein and interacts with the target of tetracycline, 70S ribosome, and promotes release of the tetracycline [28, 59]{Connell, 2003 #43;Connell, 2003 #44}. These ribosomal protection proteins have been found on transferable plasmid in C. jejuni and on mobile genetic elements in Streptomyces, making spread of this gene among bacteria possible [59].

4.4.4. Erythromycin resistanceResistance to erythromycin has been found in all Anaerostipes strains and in all Bacteroides strains (B. dorei) and (B. thetaiotaomicron). According to ARDB, erythromycin resistance can be induced in Bacteroides strains by ermf, ermg, mefa and marC genes, depending on the specific strain [38, 44, 66].

Distribution and spread of erm genes among Bacteroides speciesDuring the years, Bacteroides strains carrying ermf or ermg has been increasing since the 1970’s [80]. These erm genes cause macrolide-lincosamide-streptogramin B resistance genes and also confer resistance to clindamycin [43]. Clindamycin resistance has not been tested, but other antibiotics tested like streptomycin had the same resistant phenotype compared to erythromycin.

One study (Shoemaker et al. 2001) has investigated horizontal gene events in Bacteroides strains, isolated from both clinical and community sources, for erm genes [80]. Detection of the genes by southern blotting revealed nucleotide identity above 80%, suggesting that horizontal gene transfer was responsible for the distribution of the same genes among these related strains [80].

Other reasons to believe erm genes have been transferred horizontally between Bacteroides strains is that ermf gene has been found on transposons and conjugative transposons and has a virtual identical sequence (>99%) [43, 80]. Erm genes have been found on several conjugative transposons of the so-called CTnDOT group and on three transmissible plasmids [38, 60, 80].

mefA has a different mechanism of resistance compared to Erm genes and affects different antibiotics resistance genes [66]. The mechanism of mefA is an ATP-dependent efflux pump, keeping the concentration of erythromycin low inside the bacterial cell [66]. The mefA gene induces resistance for 14 and 15-membered macrolides, but not for 16-membered macrolides, lincosamides and streptogramin B [66]. So Anaerostipes that showed resistance for erythromycin, but not for clindamycin might possess the mefA gene or a similar gene, instead of erm genes.

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Erythromycin resistance amond AnaerostipesThe Anaerostipes isolates were all resistant to erythromycin, but not to clindamycin. Erm genes often mediate resistance for both erythromycin and clindamycin, and for this reason it is plausible that Anaerostipes do not have erm genes [44]. Other genes that can induce resistance to erythromycin are mef genes [61, 66, 81].

4.4.5. Resistance for streptomycin and kanamycinResistance for aminoglycosides kanamycin and streptomycin has been seen for all isolates, Anaerostipes and Bacteroides.

Streptomycin resistance among BacteroidesResistance to streptomycin might be caused by several genes inducing different kind of mechanisms, in Bacteroides. One mechanism is anti6ia and this is a gene found on transposons in Bacteroides which causes resistance to streptomycin [38]. Another mechanism of streptomycin resistance might also be caused by other genes and proteins like efflux pumps , (mefA) or erm genes [43, 60, 66]. These genes have been discussed in the section about erythromycin resistance in Bacteroides. More mechanisms which induce resistance to aminoglycosides exist, which are ribosomal alterations and loss of permeability [82]. Loss of permeability has also been causing resistance to cephalosporins and aminopenicillins, like ampicillin, kanamycin and cefoxitin [11, 36].

Kanamycin resistance among BacteroidesKanamycin resistance has been found in all isolated from patient 120 except for 120E BMIA2 (Parabacteroides). For patient I was also kanamycin resistance found. Resistance for kanamycin can be induced when the Bacteroides possesses the so-called aphiii3 gene [83]. This gene encodes the synthesis of enzymes which modifies the antibiotics [83].

Aminoglycoside resistance among AnaerostipesFor Anaerostipes no resistant genes responsible for aminoglycosides has been identifed before. However, the anti6ia gene has been found in many bacterial species like, enterococci and Staphylococcus aureus [41]. In the study by Kobayashi (Kobayashi et al. 2001), they mention that bacteria having both resistant genes ant6ia with aac6aph2 causes resistance for aminoglycosides and β-lactams as well. The strains possessing both genes were also resistant for ampicillin, but the mechanism behind this was not clear [41]. All aminoglycoside resistance genes originated from staphylococci, but all of these genes have been found in enterococci as well, indicating this resistance is spreading [41]. Both genes are not known to be present in Anaerostipes, but the article above makes it not unlikely that the genes are and will be transferred to Anaerostipes species as well.

4.4.6. Bacitracin resistance: AnaerostipesBacitracin was only tested in Anaerostipes. Resistance to bacitracin has been found in other bacterial strains as well. Bacitracin is obtained from Bacillus licheniformis [47]. Not only Bacillus is resistance for this agent, but also streptococcus and enterococci have shown resistance for bacitracin. Bacitracin resistance in Anaerostipes is induced by overproduction of an enzyme called undecaprenol kinase [47]. Overproduction of this enzyme up regulates the transport and synthesis of the cell wall [47]. Resistance has been found in the samples taken in the earlier phase of the treatment, suggesting the Anaerostipes lose this resistance gene during the antibiotic treatment. But this treatment should not affect Anaerostipes as all antibiotic administrated does not target anaerobic

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bacteria, or the bacteria were resistant to cephalosporins. It might be that not all Anaerostipes strains can resist the same concentration of antibiotics administered.

4.4.7. Vancomycin resistance: AnaerostipesFor vancomycin, this agent only affects Gram-positive bacteria. Anaerostipes are Gram-positive in the exponential phase, but Gram-negative in the stationary phase. Two of the strains were tolerant for vancomycin. This means the strains were inhibited by vancomycin, but not killed by the compound [49]. Tolerance for vancomcyin has been found in other bacteria as well, like staphylococcus, enterococcus and Clostridium species [49, 77]. In the article of Rose et al. 2012, they found that the methicillin-resistant Staphylococcus aureus induces tolerance after prolonged exposure of the compound. This tolerance was induced by alterations of the glycopeptides of the cell membrane [77]. It might be that Anaerostipes induce tolerance in the same way.

4.1.1. Resistance for ampicillin: BacteroidesOnly two Bacteroides isolated were resistant to ampicillin, which were isolate 120C BM IA2 (B.dorei) and IA FAA (B.thetaiotaomicron). In line with this result, many articles mentioned high resistance rates for ampicillin in Bacteroides strains [19, 30, 35-37, 84]. Two of these articles collected data-sets about susceptibility testing and rates of Bacteroides done over the years [19, 84]. The CLSI and EUCAST resistance breakpoints for ampicillin resistance rates for the Bacteroides fragilis group has been 98-100% [19]. Even worse, 45% of the strains has been highly resistant with breakpoints of 64 mg/L [19]. These high resistant levels raise the expectation that more of our Bacteroides isolates should be resistant to ampicillin as well. On the other hand in the database of ARDB genes inducing resistance to ampicillin has been found for B.fragilis, but not for B.dorei or B.thetaiotaomicron. Combining the ARDB data with our results, suggest resistance to ampicillin is not widespread among Bacteroides species.

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4.2. Plasmid transformation of E.coliPlasmids were isolated from a variety of Bacteroides and Anaerostipes strains as can be seen in the results. Transformation was succesfull for two of the plasmids. Furthermore, antibiotic resistance profiling of these transformation E.coli cells showed resistance for erythromycin. For this reason the genes responsible to erythromycin resistance are probably present on the plasmid. Concluding, the transformation experiments performed with competent E.coli cells support the idea antibiotic resistance can be spread through the microbial community, between anaerobes and aerobes.

However, the transformation experiments can be improved in many different ways. For example, horizontal gene transfer between bacterial species can be done by transformation and by conjugation. One of the alternative strategies to test horizontal gene transfer can be by performing conjugation experiments. Another reason to try conjugation experiments is that conjugation is the most common method used for exchanging genetic information [6].

Another problem occured with plasmid isolation was that not all broth dilution tests with antibiotics showed growth, in contrast to the agar dilution tests. One of reasons might be that it was difficult to add to correct quantity of antibiotic to the liquid medium, due to the small amounts needed to add (2 µl to 10 ml medium). Another possibility why in some cases plasmid transformation failed was the large sizes of the plasmid, which was above 10.000 bp. To solve this problem the plasmids can be digested with resistriction enzymes and to transform the smaller fragments in competent cells. The problem with this strategy is the unknown position of the resistant genes on the plasmid, risking cutting of the resistant gene or insertion of fragments that contain no resistant gene in the competent cells.

4.3. Conclusions: Clinical failures involving BacteroidesThe clinical failures described below illustrate the need to keep continuing with susceptibility testing of both aerobic and anaerobic bacteria. Despite the difficulties arising during testing anaerobes, these type of bacteria are often involved in bacterial infections. Furthermore, anaerobic pathogens with multiple resistant genes are increasing in number and the expectation is they need to be more often treated for infections in the future.

The first example of a multidrug resistance B.fragilis have been detected in several cases within Europe and outside leading to therapeutic failures [48]. One of these multidrug B.fragilis strains has been reported in Scandinavia, which was resistance to metronidazole, meropenem, imipenem, piperacillin-tazobactam and clindamycin [48]. The only antibiotic which affected the strain was tigecycline [48].

Another example of multiple resistant B.fragilis was from Afghanistan [48]. This strains was resistant to ampicillin/ tazobactam, clindamycin, metronidazole, cefoxitin, imipenem , meropenem, tigecycline and chloramphenicol and was only susceptible to moxifloxacin and linezolid [48].

The third example describes one multiple resistant bacteria which was already described in the introduction and was from Wareham. The B.fragilis strain was isolated in the United Kingdom and was resistant to metronidazole, β-lactams and β-lactam/β-lactamase inhibitor combinations (amoxicillin/clavulanate, piperacillin/tazobactam), carbapenems (imipenem and meropenem), macrolides and tetracyclines, penicillin and chloramphenicol [72]. This resistant strains possessed

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many different antibiotic resistant genes; cfiA (β-lactam resistant gene), ermF (MLS resistant gene), tetQ2 and tetQ3 (tetracyline resistance), qyrA (quinolone resistant region), bmeB9 and bmeB15 (efflux pump genes) were all found in this single strain [72].In our project Bacteroides strains which possibly possess the cfiA, ermF and tetQ gene has been found, indicating the antibiotic resistant genes are spread at least around Europe [72].

Multiple resistance in anaerobic gut microbiotaConcluding, the presence of multiple resistant anaerobic bacteria in the gut has been demonstrated in this study. All isolates in this study were resistant to at least two antibiotics we tested. At the ICU, the antibiotic treatment likely contributes to this effect as well, which has been shown in resistance towards the antibiotic given during treatment (cephalosporins). Furthermore, Parabacteroides and Odoribacter were sensible to the cephalosporins tested, thus administration of this agent effects these bacteria. These bacteria were less often isolated from feaces suggesting their numbers have been reduced during treatment.

But, this does not fully explain the resistance rates found for the other antibiotics e.a tetracyclin, erythromycin, streptomycin, ampicillin and kanamcyin. Resistance for these agents were present in all samples analysed during the entire antibiotic treatment, suggesting these genes were present in the bacteria before the treatment has started.

The suspicion is that horizontal gene transfer has played an important role in the multi drug resistant seen in the gut microbiota. This process is described in more detail below and also illustrated in figure 21. Therapeutic use of antibiotics in animal industry and in clinical settings led to the spread of these antibiotics in the environment, potentially contaminate food, fresh water sources and consequently end up in the human intestines [31]. This is one of the theories how gut microbes in humans who never received antibiotic treatment, carry antibiotic resistant genes with them [31]. This also supports the theory the gut plays a role as a reservoir for antibiotic resistant genes.

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Figure 21. Spread of antibiotic resistant genes throughout the environment. Therapeutic use of antibiotics led to antibiotic resistant bacteria in both humans and animals. Feacal material containing remnants of antibiotic from animals contaminate soil and fresh water. Water contaminated with antibiotics can end up in the food chain or can be used as drinking water by both humans and animals. Adapted from Kazimierczak [31].

Concluding, antibiotic treatment in clinical and industrial settings led to multiple resistance in anaerobic bacteria present in the gut. The spread and maintenance of these genes is mainly done by horizontal gene transfer involving genetic mobile elements like plasmids and transposons.

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4.4. Trouble shooting in generalFor some articles used in this discussion, it was not clear which bacteria species were included or not. Including for example Odoribacter splanchnicus, which is an ex-Bacteroides splanchnicus might led to lower resistance rates and possible underestimation [48]. Other troubles with this project was the lack of MIC’s for some of the agar diffusion tests which were performed. For Anaerostipes species were no MIC’s available at all for any test, so values from EUCAST from Gram-positive bacteria were used.

4.4.8. Trouble shooting AnaerostipesAntibiotic resistant profiling of Anaerostipes brought some difficulties. One of the problems was the quantity of information available about Anaerostipes in general but also about the specific species [23, 25, 74]. Anaerostipes rhamnosivorans has been a recently described micro-organism. Only one article (Bui et al. 2013) has been published about this species, which dates from 2013 [25]. The complete genome has not been sequenced yet, making it also not possible to screen the genome to identify chromosomal resistance genes [25]. The lack of available information has as a consequence that no clinical failures in which Anaerostipes were involved have been described.

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5. Appendix

5.1. Anaerobic culturing techniques of BacteroidesBM liquid and plates were made for growing the isolated Bacteroides. BM liquid consist of the following ingredients listed in the (table 22) below.

Table 21. BM liquid medium

Component Quantity (per litre)Brain Heart Infusion (BD) 37 gYeast extract (BD) 5 gL-cystein (sigma-aldrich) 0.5 gHeamin Bioextra from Porcine(5 g/L) (sigma-aldrich) 2.5 mlVit K1 (1mg/L) (sigma-aldrich) 0.255 mlAgar (Oxoid) 15 gResazurin (500 mg/L) 2 mldH2O Up to 1L Bottles were flushed with N2 and with N2H2. After autoclaving haemin and vitamin K1 was added. Plates with BM medium were poured into an anaerobic chamber with N2 and 2% H2.

After autoclaving the broth the medium was cooled down to 50°C before the following components were added (table 23).

Table 22. Haemin and VitK1 added to BM, RCA and MH medium.

Components Quantity (per litre)Vitamin K (0.1 ml in 20 ml) 2 mlHaemin solution (0.5 mg/ml) 10 ml

5.2. Anaerobic culturing techniques of AnaerostipesFor Anaerostipes Reinforced Clostridial agar medium (RCA) was prepared. The table below shows the composition of RCA medium per litre (table 24).

Table 23. Components for RCA medium

Components Quantity (per litre)Reinforced clostridial medium (Oxoid)

26 gr

L-cystein (sigma-aldrich) 0.5 gHeamin Bioextra from Porcine(5 g/L) (sigma-aldrich)

2.5 ml

Vit K1 (1mg/L) (sigma-aldrich) 0.255 mlAgar (Oxoid) 15 gResazurin (500 mg/L) 2 mldH2O Up to 1 L

5.3. Gram staining protocol

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A slide of cell sample was heat fixed by a flame and crystal violet was added on the slide. After the primary staining the slide was washed with water. Iodine was added to the cell sample to fix the primary stain. The primary staining that has not fixed to the cell sample was rinsed with ethanol, before the second stain (safranin) was added to the cell sample. After the final washing step with water the slide can be examined under the microscope. Gram- negative cells are pink coloured and Gram-positive cells are purple.

5.4. DNA extraction The DNA solution was transferred to an Eppendorf tube containing ALT buffer (180 µl) and proteinase K (15 µl) and incubated at 56 °C for one hour. DNase free RNase (2 µl) was added and again incubated for 15 minutes at 37 °C. After the second incubation step was AL buffer (200 µl) added and incubated for 10 minutes at 70 °C. Ethanol (200 µl) was added and the mixture was transferred to a QIAmp Minispin Column.

5.5. DNA purificationThe Qiagen column was washed twice ( 1 minute, 8000 rpm) by adding Buffer AW1 (500 µl). after the second centrifugal step was Buffer AW2 (500 µl) added. The column was centrifuged twice, the first time 3 minutes at 14000 rpm and the second time 1 minutes at 14000 rpm. The column was transferred to a fresh micro-centrifuge tube, AE buffer (50 µl) was added and the column was incubated for 5 minutes at room temperature. The final step contains a centrifuge step for 1 minute at 8000 rpm to collect the purified DNA into the tube.

5.6. Motility testingA few droplets of culture was pipetted on a clean and scratch free glass slide. The culture was covered with a cover slip and the preparation was examined immediately under the microscope. To proof motility, there was looked for directional movement that was several times longer than the long dimension of the bacterium and if this movement occurred in different directions. When all objectives were moving in a straight line in one direction this was interpreted as water current movements. Vibrational movements instead of directional were interpreted as being caused by the Brownian movement.

5.7. API test AnaerostipesFresh bacterial liquid cultures were diluted to McFarland 2 in to API 50 CHL medium. The medium was added and used to fill the API CH tubes and covered with mineral oil to create an anaerobic condition. After 24 hours and 48 hours the results were checked. The colour of the medium turns from purple into yellow if acidification of the sugars occurs and can be considers as a positive result. In case of fermentation of the sugars the colour turns into blue. To identify which bacteria were isolated our results were compared with previously described results from literature.

The API test for only performed for isolates identified as Anaerostipes. Pictures of the results were given below (figure 22).

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Figure 22.Pictures taken of API test Anaerostipes. The phenotype is shown and isolate number is given. Yellow colour indicates acidification of the sugar source and is interpreted as a positive result.

5.7.1. API test: tables of the results and identificationTable 24. Results of the API test for Anaerostipes. Sugar acidification was considered as a positive result (+), no fermentation is considered as negative result (-). In some cases the colour of the sloths was light blue or green which is considered as not clear (?).

Sugars tested

120B FAA AT

120C FAA AT 2

120 D FAA AT 1

120E FAA AT

120F RCA TC 2A

120F RCA TC 2B

Sugars tested

A. rhamnosivorans [25]

A. caccae [24]

negative ? - - - - - negative - -

glycerol + - ? ? - ? glycerol - -

Erythritol + - + + - + Erythritol - +

D-arabinose + ? + + - +D-arabinose ? +

L-arabinose + - + + - +L-arabinose - -

ribose + - + + - + ribose ? +

D-xylose ? - ? + - + D-xylose + -

L-xylose ? - - - - - L-xylose + -

Adonitol + ? + + - + Adonitol + +

b-methyl-D-xyloside - - - - - -

b-methyl-D-xyloside - -

Galactose + + + + - + Galactose + +

D-glucose + + + + - + D-glucose + +

D-fructose + + + + - + D-fructose + +

D-mannose + + + + - +D-mannose + +

L-sorbose + + + + - + L-sorbose + +

Rhamnose + + + + - + Rhamnos + -

82

e

Dulcitol + + + + - + Dulcitol + +

Inositol + + ? + - + Inositol + +

Mannitol + + + + + + Mannitol + +

Sorbitol + + + + - + Sorbitol + +

a-methyl-D-mannoside + - - - - -

a-methyl-D-mannoside - -

α methyl-D-glucoside + + + + - +

α methyl-D-glucoside + +

N-acetyl-glucosamide + + + + - +

N-acetyl-glucosamide + +

Amygdalin ? - - - - -Amygdalin - -

Arbutin + - - - - - Arbutin -variable

Esculin ferric citrate + ? ? ? ? ?

Esculin ferric citrate -

variable

Salicin ? - - - - - Salicin -variable

Cellobiose ? - - - - - Cellobiose - -

Maltose + + + + - + Maltose + +

D-lactose ? - - - - - D-lactose - -

Melibiose + + + + - + Melibiose - +

Sucrose + + + + - + Sucrose - +

Trehalose + + + + - + Trehalose + -

Inulin + - - - - - Inulin - -

Melezitose ? - - - - -Melezitose - -

D-raffinose + + + + - +D-raffinose - +

Amidon/starch ? - - - - -

Amidon/starch - +

Glycogen ? - - ? - - Glycogen - -

Xylitol + - - - - - Xylitol + +

Gentiobiose ? - - - - -Gentiobiose - -

D-turanose + + + + - +D-turanose + +

D-lyxose + ? ? ? - ? D-lyxose + +

D-tagatose + + + + - +D-tagatose + +

D-fucose + - - - - - D-fucose - -

L-fucose ? - - - - - L-fucose - -

D-arabitol + + + + - + D-arabitol + +

L-arabitol + + + + ? + L-arabitol + +

Gluconate ? - - - - - Gluconate - -2-keto gluconate - - - ? - -

2-keto gluconate -

variable

5- keto gluconate ? - - - - -

5- keto gluconate - -

Matches A. caccae 30 37 38 38 21 38Matches A. rhamnosivorans 24 40 35 36 25 38

5.8. Results disc diffusion test: Pictures Anaerostipes

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Below are pictures of the agar diffusion test of the different isolates, showing the inhibition zones for each isolate (figure 23 and figure 24).

Figure 23. Pictures of the agar diffusion test of each isolate. 1A shows the inhibition zone of isolate 120B FAA AT, 2A shows the inhibition zones of 120C FAA AT 2 and 3A shows the inhibition zones of 120D FAA AT 1.

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Figure 24. Disc diffusions test performed with Anaerostipes isolates showing the inhibition zones. 4A shows the isolate 120 E BM IA1, 5A shows isolate 120E FAA AT, 6A shows isolate 120F RCA TC A2 and 7A shows isolate 120F RCA TC 2B.

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5.9. Pictures of the disc diffusion test: Bacteroides.Below are pictures of the agar diffusion test of the different isolates, showing the inhibition zones for each isolate (figure 25).

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Figure 25. Bacteroides isolates were tested with disc diffusion test. Isolates tested were 1B:120C BMIA2, 2B: 120D FAA AT2, 3B: 120E BMIA2, 4B: IAFAA, 5B: IB3B. Antibiotics tested and abbreviations: ampicillin (AMP), tetracyclin (TET), imipenem (IPM), cefoxitin (FOX).

5.10. Preparing non-selective (Mueller Hinton) medium for antibiotic resistant profiling

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Müeller Hinton medium (MH) with agar was prepared. Antibiotic resistant profiles were investigated by adding antibiotics in proper concentrations to MH medium. The quantity of the medium per litre is shown in the table below (table 26).

Table 25. Ingredients MH medium per litre.Components Quantity (per litre)Müeller Hinton (BBL) 22 gL-cystein (sigma-aldrich) 0.5 gHeamin Bioextra from Porcine(5 g/L) (sigma-aldrich) 2.5 mlVit K1 (1mg/L) (sigma-aldrich) 0.255 mlAgar (Oxoid) 15 gResazurin (500 mg/L) 2 mldH2O Up to 1 L

5.11. Preparing non-selective (Wilkins Chalgren) medium for antibiotic resistant profiling

Wilkins-Chalgren anaerobe broth (WC) with agar was prepared. Antibiotic resistant profiles were investigated by adding antibiotics in proper concentrations to MH medium. The quantity of the medium per litre is shown in the table below (table 27).

Table 26. Ingredients WC medium per litre.Components Quantity (per litre)Wilkins-Chalgren anaerobe broth (Oxoid) 33 gL-cystein (sigma-aldrich) 0.5 gHeamin Bioextra from Porcine(5 g/L) (sigma-aldrich) 2.5 mlVit K1 (1mg/L) (sigma-aldrich) 0.255 mlAgar (Oxoid) 15 gResazurin (500 mg/L) 2 mldH2O Up to 1 L

5.12. 16S rRNA gene PCR16S rRNA PCR was performed to verify the identification of the bacteria. The table below (table 28) shows the scheme for the PCR reaction mixture.

Table 27. Components of PCR mastermix.Component Quantity per reaction (µl)10x PCR buffer + MgCl2 5dNTP mix 1Bac0027F (10x dil.) 1Uni1492R (10x dil.) 1Fast start Taq 0.4PCR H2O 40.6The colony suspension was incubated at 95 °C for 20 minutes before added to the PCR reaction mixture (1 µl). The scheme below shows the PCR programme (table 29).

Primer sequences used for 16S rRNA PCR:

5’ 27F sequence: AGA-GTT-TGA-TCM-MTG-GCT-CAG 3’ 1492R sequence: GGT-TAC-CTT-GTT-ACG-ACT-T

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M: A or C

Table 28. PCR programme used for 16S rRNA PCR.Step Temperature (°C) Time (min) Nr. Of CyclesInitial denaturation 95 5 1Denaturation 95 30 sec 35Annealing 52 40 sec 35Extension 72 1:30 35Final extension 72 10 1Soaking 4 ∞ 1

5.13. Colony PCRColony PCR was prepared to obtain 16s rRNA PCR for sequencing of the bacteria. This sequencing is needed to identify the bacteria species.

One single colony of bacteria is suspended in 100 µl of TE buffer. The single colony was treated for 10 minutes at 95 °C. After that were the tubes centrifuged for 3 minutes at 13.000 rpm. The template was only taken from the supernatant. The mastermix per reaction and PCR program are given in table 30 and 31 respectively.

Table 29. Mastermix prepared per PCR reaction.Component Quantity per reaction (µl)10x PCR buffer + MgCl2 5dNTP mix 1Bac0027F (10x dil.) 1Uni1492R (10x dil.) 1Fast start Taq 0.4PCR H2O 40.6

Table 30. Programme used for amplification of the DNA.Step Temperature (°C) Time (min) Nr. Of CyclesInitial denaturation 95 5 1Denaturation 95 1:00 35Annealing 54 1:30 35Extension 72 1:00 35Final extension 72 5:00 1Soaking 4 ∞ 1

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5.14. Preparing McFarland StandardsMcFarland standards were used to standardize the approximate number of bacteria in a liquid suspension by virtually comparing the turbidity of the bacterial suspension with the McFarland standards. Two reagents were prepared. Reagents A consist of sulfuric acid, 1% (vol/vol) and reagents B consists of Barium chloride, 1.175% (wt/vol).

The following McFarland Standards were prepared (table 32).

Table 31. Dilutions made for McFarland standards.Standard No. (McFarland) BaCl2.2H2O 1.175% (ml) H2SO4 1% (ml) OD600 nm0.5 0.025 4.975 0.091 0.05 4.95 0.2572 0.1 4.9 0.451

5.15. Phosphate buffersPhosphate buffers (100 ml of 0.1M KH2PO4 for 1L) were needed for preparation of antibiotics. Phosphate buffers with a pH of 6.0, 7.2 and 8.0 were prepared with 1M NaOH solution. After adjusting the pH the volume was completed with distilled water.

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5.16. Agar and broth dilution methodFor plates and medium containing antibiotic the following concentrations were used (table 33).

Table 32. antibiotics used for agar diffusion method.Antibiotic Antibiotic stock

concentrationEnd concentration

Diluted in? Bacteria tested?

Ampicillin natrium salz (ROTH)

10 mg/ml 100 µg/ml Phosphate buffer 0.1M pH 8.0

AnaerostipesBacteroides

Bacitracin (Sigma-aldrich) 50 mg/ml 250 ug/ml Distilled water AnaerostipesCefotoxitin (Sigma-aldrich) 20 mg/ml 5 µg/ml Distilled water Anaerostipes

BacteroidesChloroampenicol (Sigma-aldrich)

50 mg/ml 12.5 µg/ml 100% ethanol AnaerostipesBacteroides

Erythromycin (Fisher scientific)

50 mg/ml 100 µg/ml 100% ethanol AnaerostipesBacteroides

Imipenem (provided by supervisor)

20 ug/ml 20 ug/ml Phosphate buffer 0.1M pH 7.2

AnaerostipesBacteroides

Kanamycin sulfate (acros organics)

50 mg/ml 50 µg/ml Distilled water AnaerostipesBacteroides

Tetracyclin hydrochloride (ROTH)

50 mg/ml 10 µg/ml Distilled water AnaerostipesBacteroides

Streptomycin sulfate salt (sigma-aldrich)

50 mg/ml 25 ug/ml Distilled water AnaerostipesBacteroides

Vancomycin hydrochloride (sigma-aldrich)

50 mg/ml 10 ug/ml Distilled water AnaerostipesBacteroides

5.17. -lactamase disk testβSingle colonies will be diluted in PBS (Ph 7.2) untill 0.5 mcFarland. A cotton sterile swab was streaked on the entire MH plate in three different directions. The discs (table 34) are put onto the plate with a sterile tweezer, according to the scheme. After adding all discs the plate incubated in anaerobic condiitons at 37°C for 24 hrs.

Table 33. Antibiotics tested during the β-lactamase disk test.Antibiotic tested (Oxoid) Concentration disk (ug/ml) Abbreviation on diskCeftazidine 30 CAZAmoxycillin/clavulanic acid 30 AMCPiperacillin/ tazobactam 110 TZPCefotaxime 30 CTXImipenem 10 IPMMerpenem 10 MEM

Below is the scheme in which order the disks are placed onto the plates (figure 26).

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Figure 26. Scheme of order of the β-lactamase on the medium.

Interpretation is given in the table below (table 35).

Table 34. Interpretation of the β-lactamase disc test.Β-lactamase Resistance antibioticsExtended β-lactamases (ESBL’s)

Ceftazidime(CAZ) resistance [56].

ESBL’s: CTX-M types Resistance to clavulanate (AMC) and synergy towards cefotaxime (CTX) [56].ESBL’s: TEM/SHV types Resistance to clavulanate (AMC) and synergy towards ceftazidime (CAZ) [56].AmpC phenotype Resistance to ceftazidime (CAZ), but susceptible for imipenem (IMP) [73].Carbapenems Resistance to meropenem (MEM) and synergy towards piperacillin/

tazobactam (TZP) [33, 70].Carbapenems: Metallo β-lactams (MBL’s)

Resistance to all but aztreonam. Also β-lactam-β-lactamase inhibitor combinations cause resistance. Resistance to imipenem (IMP), meropenem (MEM) and piperacillin/ tazobactam (TZP) [70, 73].

5.18. CCMB80 bufferCCMB80 buffer was needed to prepare competent E.coli cells. Below is a table with the components and quantity needed (table 36).

Table 35. components needed for CCMB80 buffer.Component Concentration Quantity (g/L)KOAc (pH7.0) 10 mM 10 ml (1M stock)CaCL2.2H2O 80 mM 11.8MnCl2.4H2O 20 mM 4MgCl2.6H2O 10 mM 2Glycerol 10% 100 ml

All ingredients but MnCl2.4H2O was added to dH2O. To avoid precipitation of manganese dioxide the Ph was adjusted to 6.4 before MnCl2.4H2O was added. The CCMB media was filter sterilized and stored at 4°C.

5.19. Preparing competent cellsAfter inoculating E.coli JM109 in solid SOB media, single colonies were grown on 100 ml liquid SOB medium at 37°C, 120rpm until a density of 0.45 (OD600 nm, Ultrospec 10, Amersham Biosciences) was reached. After the culture was cooled down to 4°C the cells were washed (10 minutes, 4000 rpm) three times with cold (4°C) CCMB80 buffer. The first time 25 ml cold CCMB80 buffer solution was added, the second time 12.5 ml and the third time 5 ml. After that the cells can be stored into

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aliquots at -80°C. The efficiency of this protocol and quantity of competent cells was determined by growing competent cells of LB agar plates and count the amount of colony forming units.

5.20. TransformationPlasmids were added to the cold competent cells and transformation was performed by given the cells a heat-shock for 45 seconds at 42 °C. After the heat-shock, cells were first grown in SOC medium (1.5 hrs) and thereafter plated on LB plates with the proper antibiotic.

5.21. LB (agar) mediumAfter transformation E.coli JM 109 cells are plated on petri dishes containing LB (table 37). Below are the ingredients needed for making this medium. For solid plates agar is added as well.

Table 36. Below shows the components and quantities needed for LB medium.Component Quantity (g/L)Tryptone 10NaCl 10Yeast extract 5Agar 15After autoclaving the medium was cooled down to 50°C before antibiotics were added. Plates with antibiotics were also cooled down to 50 °C before added (final conc. 100 µg/ml). Plates were stored at 4° up to one month. Plates with Ampicillin were stored up to 4 days.

5.22. SOB and SOC (agar) mediumAfter transformation of cells with heat-shock, the cells were incubated into SOB or SOC medium. The contents of this medium (1 L) are shown in the table below (table 38).

Table 37. Components needed for SOB and SOC agar medium.Component Quantity (g/L)Tryptone 20Yeast extract 5NaCL 0.5KCl 0.186MgSO4 2.4dH2O Up to 950 mlAgar (optional) 15After adding all ingredients the pH was adjusted to 7.5. For SOC was 20 ml of 1M glucose added.

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5.23. Plasmid isolationIn short, liquid medium with fresh bacteria were prepared and grown overnight. The next day, the bacteria were harvested by centrifugation. For Gram positive bacteria (Anaerostipes), 250 µl resuspension solution was added to the pellet and 100 µl of 20 mg/ml lysozyme (from chicken egg white, Sigma Aldrich) and incubated for 20-40 minutes at 37°C. For Gram negative bacteria the lysozyme was not added. After incubation 250 µl of lysis solution was added. After mixing 350 µl neutralization solution was added. After centrifuging (14.000 rpm), the supernatant was transferred to a GeneJet spin column. The spin column was washed twice with 500 µl wash buffer. Elution buffer was heated to 70°C before applying to the silica membrane to elute plasmid DNA.

5.24. Plasmid digestion with Alu1 Isolated plasmids were digested with Alu1. Digestion has to be carried out at 37°C for 5 minutes. The enzymatic reaction was stopped by enzyme inactivation at 65° for 15 minutes. Below in table 39 the reaction scheme can be found.

Table 38. Mixture made for restriction enzyme analysis.Component Quantity (µl)Buffer B 2BSA 0.2DNA 250 ngAlu1 10U/µl (10x dil.) 0.3Nuclease free H2O Up to 20

To confirm a successful digestion a gel was run. The size of the gel pieces can be compared with the size of the plasmid.

5.25. Agarose gel electrophoresisAgarose gels of 1% and 1.5% (v/v) were made to separate DNA molecules based on size. 0.5 gr of agarose was added to 50 ml of 0.5 X TAE. The agarose was dissolved by heating with help of a microwave. The agarose was cooled down to 45°C and 10 µl of SYBR®Safe (life technologies) was added to the gel. The agarose was poured into a gel tray with flat combs. After solidifying of the gel the DNA sample were loaded in the gel. Three different DNA marker ladders were used during electrophoresis (100 mb/1kb and 1kb+). The gel was run for around 30 minutes at 100 voltage. After completion of the run a picture of the gel can be made under UV light (GeneSys).

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5.26. PCR Erm genesThe DNA template was treated for 10 minutes at 95 °C. After that were the tubes centrifuged for 3 minutes at 13.000 rpm. The template was only taken from the supernatant. Below the components in the mastermix needed per reaction. For each gene tested a separate mix need to be prepared (table 40). Also the PCR steps are given (table 41).

Table 39. mastermix prepared per gene.Component Quantity per reaction (ul)5x Buffer 5dNTP mix (10 mM) 1Primer F (10pmol/ul) 2Primer R (10pmol/ul) 2Go taq DNA polymerase (5U) 0.25Nuclease free water 12.75DNA template 2 per sample

Table 40. Steps done in the PCR programme.

Step Temperature (°C) Time (min) Nr. Of CyclesInitial denaturation 95 3:00 1Denaturation 95 1:00 35Annealing 52 1:00 35Extension 72 1:00 35Final extension 72 5:00 1Soaking 4 ∞ 1

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